Process for producing aglycon by using diglycosidase and flavor-improved food containing the aglycon and converting agent to be used in the process

ABSTRACT

A physiologically active substance of aglycon type, in particular, aglycon isoflavone, can be efficiently produced, without resort to any acid/alkali treatment or fermentation and substantially without changing the physical properties of a material, by treating the material with a sufficient amount of diglycosidase for a sufficient period of time at an appropriate temperature and pH so that a physiologically active substance of glycoside type contained in the material can be converted into the physiologically active substance of aglycon type. Moreover, by using diglycosidase and/or a specific enzyme preparation, the aglycon content in a protein or protein-containing food can be increased and the flavor thereof can be improved.

TECHNICAL FIELD

The present invention relates to a process for producing an aglycon, aprocess for producing a protein having an increased aglycon content or afood containing said protein, a process for producing a flavor-improvedprotein or a food containing said protein, and a process for forming anisoflavone in a living body. The invention may be utilized, for example,for producing processed foods, health foods, dietary supplements, andmedicaments.

BACKGROUND ART

Glycosides are chemical substances wherein a saccharide is bonded to anon-saccharide part called aglycon, and are widely present in nature.

Some glycosides are known to exhibit physiological activity throughdecomposition into aglycons by a glycosidase such as glucosidaseproduced by enteric bacteria though the glycosides themselves do notexhibit the physiological activity because of their poor entericabsorption, as phytochemicals (phytogenic functional ingredients) suchas polyphenols having antioxidation action and phytoestrogens having aweak estrogenic action. Thus, in view of preventing andsymptom-alleviating effects of life-style related diseases such ascancer, arteriosclerosis, osteoporosis, and climacteric disorder, anddiseases owing to aging, attention has been focused on soybean proteinconcentrates, soybean-processed materials, and food containing thesoybean protein concentrates.

Among the phytochemicals, glucosides that contain isoflavones asaglycons as represented by the following general formula (hereinafter,sometimes referred to as isoflavone glucosides) contain aglycons such asdaidzein, genistein, glycitein, and it is revealed from cellular levelinvestigations and epidemiological surveys that they inhibit growth ofbreast cancer and prostatic cancer cells, alleviate arteriosclerosis andosteoporosis, and also alleviate climacteric disorder owing to thefemale hormone-like estrogenic action.

(wherein R₁ and R₂ each is independently selected from the groupconsisting of H, OH, and OCH₃, and R₃ is selected from the group of H,COCH₃, COCH₂COOH, and COCH₂CH₂COOH)

However, there is a possibility that a sufficient amount of glycosidasecannot be produced by enteric bacteria in elderly persons, sick persons,and antibiotics-administered patients, and also a glycosidase isdifficult to decompose glycosides modified with acetyl or malonyl groupand disaccharide or trisaccharide glycosides, so that it cannot beexpected to absorb a sufficient amount of phytochemicals contained insoybean protein concentrates and the like.

Moreover, it is avoided to take foods containing soybean-processedmaterials such as soybean protein concentrates especially in Westerncountries owing to the distinctive smell and bitterness, and thus somelimitation exists as sources of aglycon isoflavones.

Accordingly, a process for forming phytochemicals efficiently in aliving body, and an improvement of flavor of a soybean-processedmaterial rich in aglycon isoflavones, which are highly efficientlyabsorbed and capable of ingesting a sufficient amount of isoflavones bytaking small amount of them, and having less smell and bitternessdistinctive of soybean or of a food containing the soybean-processedmaterial have been desired.

On the other hand, the method for converting an isoflavone glucosideinto an aglycon isoflavone is known as described in JP-A-10-117792. Inthis method, a phytogenic protein extract is treated with an alkali toconvert a modified glucoside isoflavone into a glucoside isoflavone,which is then subjected to a treatment with glycosidase. Such atreatment is carried out because conventional glycosidase cannot actdirectly on the modified glucoside isoflavone. Thus, the method isaccompanied by the problems of requirement of two steps, enhancement ofbitterness by the alkali-treatment, change in physical properties andingredients, formation of by-products, waste liquid after thealkali-treatment, and the like. Also, glucosidase which takes charge ofa main part of the action tends to be influenced by free glucose, sothat the kind and concentration of the material used for the productionmay be limited.

Moreover, JP-A-8-214787 describes a process for converting a glucosideisoflavone into an aglycon isoflavone by fermentation using amicroorganism. However, there are possibilities of decomposing theresulting aglycon isoflavone by the microorganism and of formingunexpected by-products, and therefore many problems may arise at theactual production.

Furthermore, a method of hydrolysis with an acid such as hydrochloricacid may be a candidate, but the decomposition of proteins,phospholipids, neutral lipids, and other ingredients may occur alongwith formation of by-products because of the severe conditions.Especially, the formation of chlorinated compounds such as MCP(monochloropropanol) and DCP (dichloropropanol) whose carcinogenicityhas been reported cannot be avoided.

Therefore, an object of the invention is to provide a process forproducing a phytogenic physiologically active substance of aglycon typeefficiently without resort of any acid/alkali treatment or fermentationand substantially without changing the physical properties of amaterial. Moreover, other objects of the invention are to enhance theaglycon content in a protein or a protein-containing food by usingdiglycosidase and/or a specific enzyme preparation and to improve theflavor. These objects and other objects will be further clarified by thefollowing detailed explanations.

DISCLOSURE OF THE INVENTION

As a result of extensive studies, we have found that diglycosidasediscovered from origins of various microorganisms efficiently decomposeglycosides which are difficult to decompose by conventional glycosidaseand also acts even in a living body, and thus accomplished theinvention. Furthermore, we have found that the invention can beconducted using any diglycosidase from any source.

Namely, the invention relates to the following.

(1) A process for producing an aglycon which comprises forming anaglycon by treating, with diglycosidase, a glycoside containing acompound selected from the group consisting of phytoestrogens,polyphenols, isoflavones, biochanin A, formononetin, cumestrol, andlignans as the aglycon.

(2) The process for producing an aglycon according to claim 1, whereinthe aglycon is an isoflavone.

(3) The process for producing an aglycon as described above, wherein theglycoside containing an isoflavone as the aglycon is one or moreselected from the group consisting of daidzin, genistin, or glycitin andacetyl derivatives, succinyl derivatives, or malonyl derivativesthereof.

(4) The process for producing an aglycon as described above, wherein thediglycosidase is a glucose-tolerant one.

(5) The process for producing an aglycon as described above, wherein thediglycosidase is diglycosidase produced by Penicillium multicolorIAM7153.

(6) A process for producing a protein having an increased aglyconcontent or a food containing the protein, which comprises a step oftreating a protein or protein-containing food with diglycosidase.

(7) The process for producing a protein having an increased aglyconcontent or a food containing the protein as described above, wherein theprotein or protein-containing food contains a glycoside containing anisoflavone as the aglycon.

(8) The process for producing a protein having an increased aglyconcontent or a food containing the protein as described above, wherein theprotein or protein-containing food to be produced is a furtherflavor-improved one.

(9) The process for producing a protein having an increased aglyconcontent or a food containing the protein as described above, wherein theglycoside containing an isoflavone as the aglycon is one or moreselected from the group consisting of daidzin, genistin, or glycitin andacetyl derivatives, succinyl derivatives, or malonyl derivativesthereof.

(10) The process for producing a protein having an increased aglyconcontent or a food containing the protein as described above, whichfurther comprises a step of treating with an enzyme preparationcontaining mainly at least one enzyme selected from the group consistingof amylases, proteases, lipases, α-glucosidases, and yeast-dissolvingenzymes.

(11) The process for producing a protein having an increased aglyconcontent or a food containing the protein as described above, wherein theimprovement of flavor is reduction of bitterness and/or astringency.

(12) A process for producing a flavor-improved protein or a foodcontaining the protein, which comprises a step of treating with anenzyme preparation containing mainly at least one enzyme selected fromthe group consisting of amylases, cellulases, pectinases, proteases,lipases, α-glucosidases, α-galactosidase, and yeast-dissolving enzymes.

(13) The process for producing a flavor-improved protein or a foodcontaining the protein as described above, wherein the protein orprotein-containing food contains a glycoside containing a flavonoid asthe aglycon.

(14) The process for producing a flavor-improved protein or a foodcontaining the protein as described above, wherein the protein orprotein-containing food contains a glycoside containing an isoflavone asthe aglycon.

(15) A method of administering diglycosidase orally to form an aglyconfrom a glycoside in a living body.

(16) The method as described above, wherein diglycosidase is orallyadministered to form an aglycon in a living body from a glycosidecontaining an isoflavone as the aglycon.

(17) A method of converting a physiologically active substance ofglycoside type into a physiologically active substance of aglycon type,which comprises treating the physiologically active substance ofglycoside type with diglycosidase.

(18) A process for producing a composition rich in a phytogenicphysiologically active substance of aglycon type, which comprisestreating a phytogenic material containing a phytogenic physiologicallyactive substance of glycoside type with diglycosidase.

(19) A method of accelerating a bioabsorption of a physiologicallyactive substance, which comprises administering diglycosidase orallybefore, during, or after the ingestion of a food containing aphysiologically active substance of glycoside type.

(20) An agent converting a physiologically active substance of glycosidetype into the physiologically active substance of aglycon type, whichcontains at least diglycosidase.

These embodiments and other embodiments of the invention will be furtherclarified by the following detailed explanations.

Diglycosidase described in the invention efficiently acts on theglycosides that contain a compound selected from the group consisting ofphytoestrogens, polyphenols, isoflavones, biochanin A, formononetin,cumestrol, and lignans as the aglycon (hereinafter, also referred to asaglycon glycoside), can very efficiently act especially on theglycosides containing an isoflavone as the aglycon (hereinafter, alsoreferred to as isoflavone glycosides), and is hardly influenced by freeglucose. Therefore, the process can be advantageously carried out in thecase that the isoflavone glycoside is daidzin, genistin, or glycitin, oran acetyl derivative, succinyl derivative, or malonyl derivativethereof. By the way, the isoflavone formed from the isoflavone glycosideis also referred to as aglycon isoflavone.

Moreover, by administering the diglycosidase, the preventive effect onvarious diseases can be enhanced through the formation of an isoflavonefrom an aglycon glycoside or a food containing the same which is orallyingested. Diglycosidase produced by Penicillium multicolor IAM7153 ispreferably used as diglycosidase. β-Galactosidase derived fromPenicillium multicolor is an enzyme which is described in Food AdditivesList and whose safety is recognized, and thus diglycosidase produced bysuch highly safety bacterium is estimated to be highly safe.

Furthermore, in the case of using soybean as a starting material,although the benefit of soybean protein concentrates andsoybean-processed materials to health is reported, ingestion of them areavoided especially in Western countries owing to the distinctive smelland bitterness. The above-mentioned acetylglycoside isoflavones andmalonylglycoside isoflavones have a strong bitterness but the aglyconisoflavones have less bitterness. Therefore, a food wherein bitternessof soybean protein concentrate or soybean-processed material isefficiently reduced can be provided by the conversion into aglyconisoflavones by diglycosidase.

When such soybean protein concentrate or soybean-processed materialwherein isoflavones are concentrated as aglycons having higherabsorption efficiency is used, a sufficient amount of isoflavones can beingested through a little intake, and also the form can be changed to aform easily ingested by people who avoid the smell and strange tastedistinctive of soybean.

Moreover, owing to the smell and strange taste of soybean proteinconcentrates or soybean-processed materials, the amount used is limitedin the case of using them as food materials, and therefore soybeanprotein concentrates or soybean-processed materials have a limitation asisoflavone sources for foods. According to the invention, use of thesoybean protein concentrates or soybean-processed materials, whereinisoflavone glycosides are digested with diglycosidase and concentratedas isoflavone aglycons which have higher absorption efficiency and lessbitterness, enables supply of isoflavones to foods through a little useof the concentrates or materials, so that they can be utilized for manykinds of foods as isoflavone sources.

Diglycosidase for use in the invention is characterized in that it hasan activity of acting on a disaccharide glycoside, which is difficult toutilize as a substrate by conventional glucosidase, to isolate asaccharide as a two-saccharide unit from the disaccharide glycoside andalso to form an aglycon. Herein, an enzyme having the above activity isreferred to as “diglycosidase”.

The diglycosidase in the invention is an enzyme which is classified intoa saccharide-chain hydrolase but has a property different from theproperties of conventional α- and β-glycosidases. Diglycosidase canutilize, as a substrate, so-called a glycoside wherein a linear orbranched saccharide chain composed of single or two or more kinds ofsaccharides is bonded to a compound other than a saccharide throughhydroxyl group in the saccharide chain, and recognizes the substrate atthe two-saccharide unit to cleavage it, whereby correspondingdisaccharide and an aglycon having a saccharide chain with twosaccharide-smaller chain length are formed successively and finally, anaglycon is formed. Additionally, it also decomposes modified glucosidessuch as acetyl derivatives, succinyl derivatives, and malonylderivatives, which are difficult to decompose by conventionalglucosidase, into saccharides and aglycons. As representative examplesof saccharides present in nature, starch, cellulose, polysaccharidesconstituting cell walls, and the like may be mentioned. Many kinds ofsaccharide chains may be suitable for the saccharide chains ofglycosides, and examples thereof include6-O-β-D-xylopyranosyl-β-D-glucopyranoside (β-primeveroside),6-O-α-L-arabinopyranosyl-β-D-glucopyranoside (vicianoside),6-O-α-L-arabinofuranosyl-β-D-glucopyranoside,6-O-α-L-rhamnopyranosyl-β-D-glucopyranoside (rutinoside),6-O-β-D-apiofuranosyl-β-D-glucopyranoside,6-O-β-D-glucopyranosyl-β-D-glucopyranoside (gentiobioside),4-O-α-glucopyranosyl-β-D-glucopyranoside (maltose),2-O-α-L-rhamnopyranosyl-β-D-galactopyranoside (rhaminose),6-O-α-L-rhamnopyranosyl-β-D-galactopyranoside (robinobioside),2-O-β-D-xylopyranosyl-β-D-glucopyranoside (xylosylglucose),4-O-β-D-glucopyranosyl-β-D-glucopyranoside (cellobioside), xylobioside,and the like. Other than the above-mentioned compounds, any combinationof saccharides can be recognized as a substrate for the reaction as faras the combination has a disaccharide structure. Aglycon means acompound to be obtained from a glycoside by eliminating a saccharide ofthe glycoside. Aglycons of glycosides are widely present in nature, andexamples thereof include volatile compounds in plants such as linalool,geraniol, citronellal, phenethyl alcohol, citronellol, jasmones,limonene, terpinene, citral, nerol, pinene, borneol, terpineol, methyljasmonate, hexanol, hexenol, hexanal, hexenal, vanillin, benzaldehyde,eugenol, methyl salicylate, linalool oxide, benzyl alcohol, andvomifomitol; pigments in plants such as alizarin, purpurin,anthocyanidin including pellagonidin, cyanidin, delphinidin, peonidin,petunidin, and malvidin; and flavonoids such as nariltin, naringenin,hesperetin, neohesperetin, diosmetin, quercetin, campherol, myricetin,isorhamnetin, and syringenin; and the like. Other than the compoundsmentioned herein, various compounds may be present as aglycons ofglycosides or may become aglycons of glycosides.

Furthermore, diglycosidase can utilize so-called monosaccharideglycosides, wherein one molecule of saccharide is bonded to an aglycon,as substrates to form corresponding monosaccharides and aglycons, otherthan above-mentioned disaccharide-isolating activity. In particular, itis a characteristic that diglycosidase can act on monosaccharideglycosides which is resistant to hydrolysis by conventionalβ-glucosidase.

Diglycosidase for use in the invention can be obtained frommicroorganisms having ability of producing diglycosidase withoutrequiring undue experimental burden from those skilled in the art (Forexample, cf. WO00/18931).

The microorganisms producing diglycosidase of the invention can beobtained by the following screening, for example. That is, an enrichmentculture is carried out by inoculating a soil suspension to a liquidmedium for separation containing eugenyl primeveroside or the like assole carbon source, applying the culture liquid onto a similar platingagar medium for separation and selecting colonies grown. These strainsare cultured in a suitable liquid medium and strains havingpNP-isolating activity can be selected through cleavage of disaccharidefrom pNP-primeveroside or the like.

On these strains thus selected, microorganisms producing diglycosidasecan be screened using pNP-primeveroside or the like as a substrate anddisaccharide isolation as a measure.

The producing ability has been already confirmed on Aspergillus nigerIFO4407 (available from Institute of Fermentation, 2-17-85,Juso-honmachi, Yodogawa-ku, Osaka), Aspergillus niger IAM 2020,Aspergillus fumigatus IAM2046, Penicillium multicolor IAM7153 (availablefrom Institute of Molecular Cell Biology, the University of Tokyo,1-1-1, Yayoi, Bunkyo-ku, Tokyo), and the like.

Additionally, in other various microorganisms, the diglycosidaseactivity has been confirmed on various microorganisms such as the genusAspergillus, the genus Penicillium, the genus Rhizopus, the genusRhizomucor, the genus Talaromyces, the genus Mortierella, the genusCryptococcus, the genus Microbacterium, the genus Corynebacterium, thegenus Actinoplanes, and the like.

Any strain can be used in the invention as far as it has an ability ofproducing diglycosidase, and the strain is not limited to theabove-mentioned strains. Furthermore, the process for producingdiglycosidase usable in the invention includes mutant strains of thestrains having a diglycosidase-producing ability, or variousmicroorganisms or various cells (e.g., yeast cells, bacterial cells,higher plant cells, and animal cells) modified so as to be capable ofproducing diglycosidase by recombinant DNA method, and particularlypreferred are those modified so as to be capable of producingdiglycosidase with high productivity. In the case that adiglycosidase-producing ability is imparted by introducing adiglycosidase gene, the microorganism used as a host may not have adiglycosidase-producing ability.

For producing diglycosidase using the above various microorganisms, amethod and conditions suitable for the culture of the microorganism canbe set, and the method and conditions are not particularly limited. Forexample, any of liquid culture and solid culture may be used forculturing the above various strains, but liquid culture is preferablyused. The liquid culture may be carried out as follows, for example.

The medium to be employed may be any medium as far as the microorganismproducing diglycosidase is capable of growing in the medium. Forexample, there may be used media to which carbon sources such asglucose, sucrose, gentiobiose, soluble starch, glycerol, dextrin,molasses, and organic acids; further nitrogen sources such as ammoniumsulfate, ammonium carbonate, ammonium phosphonate, ammonium acetate, orpeptone, yeast extract, corn steep liquor, casein hydrolysate, bran, andmeat extract; and further inorganic salts such as potassium salts,magnesium salts, sodium salts, phosphonates, manganese salts, ironsalts, and zinc salts are added. Furthermore, for accumulatingdiglycosidase, various inducing substances may be added to the medium.As the inducing substances, saccharides may be used, for example, andthere may be preferably used gentose (e.g., gentose #80, Nihon ShokuhinKako Co., Ltd.), gentiobiose, genti-oligosaccharide (e.g., gentiologoetc., Wako Pure Chemical Industries, Ltd.), galactomannan, and the like.The adding amount of these inducing substances is not particularlylimited as far as the productivity of aimed diglycosidase is enhanced,but the substance is preferably added in an amount of 0.01 to 10%.

The pH of the medium is adjusted to from about 3 to 8, preferably fromabout 5 to 6, and culture is carried out at a temperature of about 10 to50° C., preferably about 25 to 30° C. for 1 to 15 days, preferably 4 to7 days under aerobic conditions. As the culturing method, a shakingculture or an aerobic submerged culture by means of a jar fermenter maybe utilized. However, the above various culturing conditions may beoptionally changed, of course, depending on the microorganism or cell tobe cultured, and the conditions are not particularly limited as far asdiglycosidase of the invention is produced.

For isolation and purification of diglycosidase from the culture liquidobtained, using a diglycosidase activity as a measure, purifieddiglycosidase can be obtained by combining centrifugal separation, UFconcentration, salting out, and various chromatography such as ionexchange resins, and treating in a usual manner (Referential document:Tanpakusitsu·Kouso no Kisojikkenhou (Basic experimental methods forproteins and enzymes), written by Takekazu Horio, Nankodo).

A culture liquid obtained by culturing the above microorganism may beutilized as such as the enzyme composition of the invention. Of course,the culture liquid may be optionally changed in the degree ofpurification according to the purpose used in the invention.

The invention provides a process for producing an aglycon whichcomprises forming an aglycon by treating, with diglycosidase, aglycoside containing a compound selected from the group consisting ofphytoestrogens, polyphenols, isoflavones, biochanin A, formononetin,cumestrol, and lignans as the aglycon. The producing process includesthe reaction of a phytogenic material containing the above compound asthe aglycon with a sufficient amount of diglycosidase under weaklyacidic conditions at an appropriate temperature and pH for a sufficientperiod of time so as to convert at least most of the glycoside in thestarting material into an aglycon, whereby an aglycon is produced. Theinvention provides a producing process wherein diglycosidase is added toa plant extract in order to produce a plant extract rich in an aglycon.

The novel process is a one-step process of converting most of an aglyconglycoside into free aglycon by an enzyme preparation containing ahydrolase of disaccharide glycosides, i.e., diglycosidase. The processis effective for the aglycon glycosides present in phytogenic materials,preferably proteins or protein foods. Since the process is found to becapable of substantially complete conversion of modified glucosideisoflavones and glucoside isoflavones into aglycon isoflavones, itincludes the conversion of modified glucoside isoflavones and glucosideisoflavones into aglycon isoflavones. In some phytogenic proteinmaterials, particularly soybean protein materials, substantial part oftotal isoflavone contents in the phytogenic protein materials is presentin the form of isoflavone glycosides. Therefore, not only the conversionof glycoside isoflavones into aglycon isoflavones but also theconversion of modified glycoside isoflavones into aglycon isoflavonesare necessary for maximum increase of the amount of aglycon isoflavonesobtainable from the phytogenic protein materials.

The starting material in a preferred embodiment is any protein orprotein-containing food (more preferably a phytogenic material,phytogenic protein, or phytogenic protein-containing food) containing aphysiologically active substance of glycoside type. Some processes inthe following explanations are described using soybean products asexamples, but the process of the invention can be generally applied to awide range of proteins or protein-containing foods other than soybeanand soybean products.

In the invention, the “protein or protein-containing food” preferablycontains a physiologically active substance of glycoside type but is notparticularly limited.

The “phytogenic material” in the invention means a whole plant bodywhich is edible or used as a medicine, or a part thereof such as leaf,flower, fruit, stem, or root, or a processed product thereof. Examplesthereof include whole plant bodies harvested, or parts thereof such asleaf, flower, fruit, stem, and root, and plant extracts and processedproducts thereof. Specific examples of the phytogenic material includethe following materials: phytogenic proteins such as soybean protein,soymilk, juices (orange juice, grape juice, apple juice, pomegranatejuice), herb tea, plant extracts such as herb extract, and processedproducts of the above materials such as juice drinks, wine, tea, blacktea, and cocoa.

The “phytogenic protein” means a protein obtainable from the above“phytogenic material”, and may be a mixture with other ingredientsderived from the phytogenic material.

In the invention, the “compound selected from the group consisting ofphytochemicals, phytoestrogens, polyphenols, isoflavone, biochanin A,formononetin, cumestrol, and lignans” is not particularly limited as faras the compound falls within these conceptual range, but it ispreferably a compound which exhibits a physiological activity orenhances a physiological activity in a living body (preferably awarm-blooded animal, more preferably human). The compound is preferablya flavonoid, more preferably an isoflavone, most preferably anisoflavone represented by the above structural formula.

The “physiologically active substance” in the invention means asubstance, most of which is preferably present as a glycoside in a plantbody and which exhibits a physiological activity or enhances aphysiological activity in a living body upon the conversion into theaglycon type. Specifically, the physiologically active substanceincludes phytochemicals, phytoestrogens, polyphenols, isoflavone,biochanin A, formononetin, cumestrol, and lignans as mentioned above,and preferred are isoflavones.

The “physiologically active substance of glycoside type” means that theaglycon of the above glycoside is a physiologically active substance,and the saccharide chain is composed of one or more saccharide,preferably two or more saccharides. The two-saccharide chains includethose mentioned above and the like.

The “enzyme preparation containing mainly at least one enzyme selectedfrom the group consisting of amylases, proteases, lipases,α-glucosidases, and yeast-dissolving enzymes” is not particularlylimited as far as it mainly contains these enzymes, and commerciallyavailable enzymes may be employed. The following will illustrates thosemanufactured by Amano Enzyme Inc. Examples of amylase include Amylase AD“Amano” 1 (optimum pH: 6.0, optimum temperature: 70° C.), Gluczyme NL4.2 (optimum pH: 4.5, optimum temperature: 65° C.), Transglucosidase L“Amano” (optimum pH: 5.0, optimum temperature: 60° C.), and the like.Examples of cellulase include Cellulase A “Amano” 3 (optimum pH: 4.5,optimum temperature: 55° C.), Cellulase T “Amano” 4, Hemicellulase“Amano” 90G (optimum pH: 4.5, optimum temperature: 50° C.),Hemicellulase GM “Amano”, and the like. Examples of pectinase includePectinase PL “Amano” (optimum pH: 4.55–0, optimum temperature: 60–55°C.) and the like. Examples of protease include Umamizyme, Newlase F3G,Papain W-40, Pancreatin F, Protease B, Protease A “Amano” G. and thelike, and examples of lipase include Lipase A “Amano” 6 (optimum pH:6.5, optimum temperature: 45° C.), and the like. A yeast-dissolvingenzyme preparation YL-15 (optimum pH: 7.0, optimum temperature: 50–55°C.) is mentioned as a yeast-dissolving enzyme, and ADG-S-DS (optimum pH:4.5–5, optimum temperature: 50–60° C.) and the like are mentioned as anα-galactosidase.

The above enzymes and enzyme preparations can be produced by knownmethods. For example, an enzyme can be obtained by screening amicroorganism producing a specific enzyme mentioned above in a similarmanner to the production of diglycosidase and culturing the resultingenzyme-producing strain in a suitable medium. Examples of the aboveenzyme-producing strain include Bacillus subtillis, Aspergillus niger,Aspergillus oxyzae, Trichoderma viride, Rhizopus nivenus, Pseudomonassp., and the like.

The following will explain the invention in further detail with regardto the process for producing a protein having an increased aglyconcontent or a food containing the protein, which comprises a step oftreating a protein or protein-containing food with diglycosidase, by wayof illustration of a phytogenic physiologically active ingredient(especially an isoflavone glycoside) derived from a phytogenic material,but the invention can be conducted using any above compound other thanthe phytogenic physiologically active ingredient derived from aphytogenic material. By the way, the term of soybean material usedherein means any type of soybean or variants of soybeans.

Some different embodiments are possible as specific processes forcarrying out the invention.

In the first embodiment, a phytogenic physiologically active substanceof glycoside type is converted into a phytogenic physiologically activesubstance of aglycon type while the phytogenic physiologically activesubstance is left in the phytogenic material. Therefore, the formedphytogenic physiologically active substance of aglycon type may be leftin the phytogenic material or may be suitably removed. The aglycon formof the phytogenic physiologically active substance may be generallyremoved by a solvent, hydrophobic effluence or extraction. The solventsuitable for the operation includes acetone, ethanol, and other similarorganic solvents, but is not limited thereto.

In the second embodiment, a phytogenic physiologically active substanceof glycoside type (e.g., isoflavone modified glycoside or isoflavoneglucoside) in a phytogenic material is removed from the phytogenicmaterial by aqueous effluence or extraction. The aqueous effluence iscarried out through the effluence of relatively soluble phytogenicphysiologically active substance of glycoside type by immersing thephytogenic material or by exposing the phytogenic material to or dippingit in water or a mixture of hydrophilic solvents such as ethanol orother alcohols. The pH of the resulting aqueous solution is from aboutpH 2 to about pH 5, preferably about pH 4. After removal, the phytogenicphysiologically active substance of glycoside type is converted into thephytogenic physiologically active substance of aglycon type.

In the third embodiment, prior to all the operations for conversion, aphytogenic physiologically active substance of glycoside type is removedfrom a phytogenic material.

Depending on the type of phytogenic material containing a phytogenicphysiologically active substance of glycoside type, in some cases, thephytogenic material is preferably processed to a finely crushed form.This operation is desirable for bringing a phytogenic physiologicallyactive substance in the phytogenic material into contact with a reagent(diglycosidase) employed in the step which will be described in detailin the following. The material may be subjected to grinding, crushing,or other processing. When the phytogenic material is in condition thatisoflavone compounds in the phytogenic material easily come into contactwith an external reagent or reactant, e.g., a small leaf part in aplant, it is not necessary to subject the phytogenic material to theabove processing.

The conversion of a phytogenic physiologically active substance ofglycoside type into the phytogenic physiologically active substance ofaglycon type is sometimes partially carried out by enzymes present inthe mixture depending on the phytogenic material used. These enzymes maybe present naturally in phytogenic protein materials or may be derivedfrom microorganisms grown in the materials. Such enzymes are called asresidual enzymes. However, there is a possibility that the conversion ofthe phytogenic physiologically active substance of glycoside type intothe phytogenic physiologically active substance of aglycon type cannotbe carried out sufficiently depending on the nature and concentration ofthe residual enzyme in the phytogenic protein materials. By adding anenzyme preparation containing an external enzyme, i.e., diglycosidase,maximum converting efficiency of the phytogenic physiologically activesubstance of aglycon type can be achieved.

In the invention, the amount of the enzyme to be added depends onvarious factors including the type of enzyme present, the distributionof enzyme concentration, the pH of reaction system, the activity ofenzyme present, and temperature. In the case of adding an enzyme,typically preferred enzyme amount is preferably from 28 to 2800 AU,usually from 10 to 10000 AU relative to 100 g of a phytogenic materialas total concentration of enzyme present based on dry weight thereof.When a sufficient concentration of enzymes including a residual enzyme,an additional enzyme or both enzymes are present in the system, aphytogenic physiologically active substance of glycoside type is broughtinto contact with the enzymes at an appropriate temperature and pH for asufficient period of time so as to convert substantially all thephytogenic physiologically active substance of glycoside type in themixture into the phytogenic physiologically active substance of aglycontype.

The conversion-production step is preferably carried out at a pH ofabout 2 to about 6. More preferred pH range for theconversion-production step is from about 3 to about 5. Depending on thephytogenic material used, the pH may be adjusted with an acidic reagentsuch as hydrochloric acid, phosphoric acid, acetic acid, or sulfuricacid, or an alkaline reagent such as sodium hydroxide. In many cases, itis assumed to use an acidic or alkaline reagent of food grade. Thetemperature to be used in the conversion-production step is preferablyfrom about 25° C. to about 65° C. More preferred temperature is fromabout 30° C. to about 55° C. Throughout the reaction, the temperature isusually constant, but the temperature may be elevated or loweredaccording to the successive step and final intended use. Namely, it maybe relatively freely changed according to the various circumstances ofthe situation.

The period of time necessary for the conversion and production may bedetermined depending on complicated relationship between various factorsof the kind, concentration, and physical properties of the material tobe reacted, the concentration of the enzyme added, and further thetemperature and pH of the reaction system. In most cases, theconversion-production can be substantially completely achieved within 6to 12 hours. The period of time for the conversion-production can beshortened depending on the concentration of the diglycosidasepreparation added. At the conversion-production step, most of theisoflavone glycoside in the mixture can be converted into the aglyconisoflavone. The efficiency of the conversion is usually at least about50% or more, preferably about 70% or more. By adopting the abovepreferable reaction conditions, nearly complete conversion can beachieved.

By adopting conditions similar to the above, it is possible to carry outa process of the invention for producing an aglycon which comprisesforming an aglycon by treating, with diglycosidase, a glycosidecontaining a compound selected from the group consisting ofphytoestrogens, polyphenols, isoflavones, biochanin A, formononetin,cumestrol, and lignans as the aglycon.

In addition to the above step, in the invention, the process may furthercomprise a step of treating with an enzyme preparation containing mainlyat least one enzyme selected from the group consisting of amylases,proteases, lipases, α-glucosidase, and yeast-dissolving enzymes. Thisstep may be conducted before or after the step of treating withdiglycosidase, or the treatment with diglycosidase and the enzymepreparation may be carried out at the same time. In this case, thetreatment with diglycosidase and the enzyme preparation at the same timeis carried out under the conditions similar to those in the case ofusing diglycosidase solely. Moreover, when the treatment with an enzymepreparation is carried out before or after the treatment withdiglycosidase, the pH, temperature, period of time, and the like may beselected in consideration of optimum pH and optimum temperature of theabove each enzyme preparation. This process is also accompanied by theeffects of increasing aglycon content in a protein or protein-containingfood and of improving flavor through the reduction of bitterness and/orastringency.

Additionally, in the invention, during the process of finding the aboveeffects of the combined use with diglycosidase, it was found that flavoris improved by treating a protein or protein-containing food with aspecific enzyme preparation alone, i.e., an enzyme preparationcontaining mainly at least one enzyme selected from the group consistingof amylases, cellulases, pectinases, proteases, lipases, α-glucosidase,α-galactosidases, and yeast-dissolving enzymes. With regard to thetreating conditions in this case, the pH is preferably from 3 to 8, morepreferably from 5 to 7.5, and the treating temperature and treating timeare similar to the case of the combined use with diglycosidase. By theway, the treated product may be optionally adjusted to a desired pH.

By treating a protein or protein-containing food as mentioned above, theflavor of the protein or protein-containing food can be improved andparticularly, bitterness and/or astringency can be reduced.

In addition, by administering the phytogenic physiologically activesubstance of aglycon type produced as above or a composition rich in thephytogenic physiologically active substance of aglycon type as such oras a mixture with a food or drink, the effect derived from thephytogenic physiologically active substance can be attained. The effectsof the phytogenic physiologically active substance include effects ofpreventing various diseases (cancer, life-style related diseases,osteoporosis, a burning sensation in climacteric disorder, and thelike), and of regulation of intestinal function, immunostimulation, andbiophylactic action. Moreover, other than the administration of thephytogenic physiologically active substance which is converted intoaglycon type beforehand, by administering orally a phytogenicphysiologically active substance of glycoside type and/or a phytogenicmaterial containing a phytogenic physiologically active substance ofglycoside type together with diglycosidase, the phytogenicphysiologically active substance of glycoside type is converted into thephytogenic physiologically active substance of aglycon type in a livingbody, for example in stomach or intestines and the absorption of thephytogenic physiologically active substance of aglycon type and themigration into blood are accelerated, whereby preventive effect of thephytogenic physiologically active substance to various diseases can beenhanced.

The method of accelerating a bioabsorption of a physiologically activesubstance according to the invention, which comprises administeringdiglycosidase orally before, during, and/or after the ingestion of afood containing physiologically active substances of glycoside type,(preferably, a method of forming an isoflavone from a glycosidecontaining an isoflavone as the aglycon in a living body), is conductedas follows.

The target is a warm-blooded animal, preferably human or livestock.

As far as the phytogenic physiologically active substance of glycosidetype comes into contact with diglycosidase in stomach and intestines,diglycosidase may be administered at any time before, during, or afterthe ingestion of a food containing physiologically active substances ofglycoside type. Preferred is between just after a meal and one hourafter the meal.

The dose of diglycosidase is not particularly limited as far as theconversion of the phytogenic physiologically active substance ofglycoside type into the phytogenic physiologically active substance ofaglycon type occurs in a living body, but diglycosidase is orallyadministered in an amount of usually from 10 mg/day to 500 mg/day,preferably from 30 mg/day to 300 mg/day, more preferably 100 mg/day to200 mg/day. The number of dose is not particularly limited but ispreferably from once per several days to several times per day,particularly preferably three times per day (i.e., after every meal).

Moreover, the ingesting amount of the physiologically active substanceis not particularly limited as far as its effect is attained, but thesubstance is ingested in an amount of preferably 10 mg/day or more, morepreferably 50 mg/day or more, further preferably 50 mg/day to 100mg/day.

Furthermore, diglycosidase may be administered solely, as an enzymepreparation, and/or as a mixture with conventional glycosidase (e.g.,glucosidase, galactosidase, etc.).

Diglycosidase may be used as an enzyme preparation. In this case, theenzyme preparation contains diglycosidase as the essential ingredient,and may further contain various enzymes, stabilizers, and the like.

Additionally, in the case of the administration as an enzyme preparationmixed with conventional glycosidase, examples of the conventionalglycosidase include glucosidase, galactosidase, xylosidase, andrhamnosidase, and the dose of diglycosidase is preferably from 10 mg/dayto 50 mg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of Example 7.

FIG. 2 is a graph showing the results of Example 8.

FIG. 3 is a graph showing the results of Example 14.

FIG. 4 is a graph showing the results of Example 15.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention will be explained in detail with illustrating Examplesusing soybean materials as phytogenic materials. Examples areillustrated for the purpose of explanation only and they by no meansrestrict the scope of the invention.

The phytogenic materials, for example defatted soybean, soymilk,concentrated soybean protein, and various soybean products contain 12kinds of isoflavone compounds. Specifically, they contain aglycons ofglycitein, daidzein, and genistein; glucoside glycosides of glycitin,daidzin, and genistin; acetylglycitin, acetyldaidzin, and acetylgenistinhaving O-acetyl group at 6-position of the glucose residue; andmalonylglycitin, malonyldaidzin, and malonylgenistin having O-malonylgroup at 6-position of the glucose residue. The existing ratio of thesecompounds is characteristic to each of the difference of varieties ofsoybean and the difference of treatment in the production steps.

Unless otherwise stated, ratio, part(s), percent, and the like areherein based on weight.

By the way, the measured activities of various enzymes herein are shownas values obtainable by the method described below unless otherwisestated.

Diglycosidase Activity

The activity was measured on an automatic chemical analyzing apparatus(TBA-30R manufactured by Toshiba Corporation). Thirty μL of an enzymesample was mixed with 200 μL of 2 mM solution of p-nitrophenyl (pNP)primeveroside used as a substrate of disaccharide glycoside, which isobtained by dissolving the compound in an acetate buffer (pH 5.5),followed by reaction at 40° C. for 9.75 minutes at the cycle time of22.5 seconds. Then, 250 μL of sodium carbonate was added thereto andthen absorbance at 412 nm was measured. A blank derived from the samplewas measured similarly using 20 mM acetate buffer (pH 5.5) instead ofthe substrate solution.

The enzyme amount increasing the absorbance by 1 under the conditions isdefined as 1 AU.

The pNP-primeveroside used herein can be synthesized, for example, byreacting pNP-glucoside (manufactured by Merck) with xylo-origosaccharide(manufactured by Wako Pure Chemical Industries, Ltd.) using an enzyme,xylosidase (manufactured by Sigma) to bond xylose to pNP-glucoside inβ-1,6-manner via one residue transfer.

EXAMPLE 1 Production of Diglycosidase by Penicillium multicolor IAM7153

Culture of Diglycosidase

A medium for growth (pH 5.6) containing 2.0% of defatted soybean, 3.0%of glucose, 0.5% of potassium dihydrogen phosphate, 0.4% of ammoniumsulfate, 0.3% of dry yeast was sterilized at 121° C. for 20 minutes. To100 mL of the sterilized medium was inoculated 1 oese of Penicilliummulticolor IAM7153, followed by pre-culture at 27° C. at the shakingrate of 140 min⁻¹. After 5 days, 20 L of a main medium of pH 4.9containing 1.0% of Sunfiber R, 2.0% of potassium dihydrogen phosphate,1.0% of ammonium sulfate, and 3.13% of meast P1G was sterilized in a 30L jar fermenter at 121° C. for 20 minutes while stirring at 150 min⁻¹.The pre-medium was inoculated at a rate of 1.5% and the whole wascultured at a stirring number of 250 min⁻¹, an aeration of 0.75 vvm (15L/min), an inner pressure of 0.5 kg/cm² (48 kPa), and a temperature of27±1° C. for 8 days.

Purification of Diglycosidase

To the culture broth were added 2% by weight each, based on total liquidamount, of Zemlite Super 56M and Fineflow A as filtration aids andfiltration through diatomaceous earth was carried out. The filtrate wasconcentrated by a factor of 20 using an ultrafiltration membrane UFAIP-2020 (MW 6,000) and also the substitution by 20 mM acetate buffer ofpH 4.7 was conducted. Ammonium sulfate was added to the aboveultrafiltration concentrate to conduct 50% ammonium sulfate-salting out.The resulting precipitate was removed and ammonium sulfate was furtheradded to the supernatant to conduct 80% ammonium sulfate-salting out.The precipitate was recovered and dissolved in 20 mM acetate buffer ofpH 4.7. The solution was passed through a 10-DG column (BioRad Co.) toexchange the buffer for 20 mM acetate buffer of pH 4.7 containing 30%saturated ammonium sulfate (this solution is also referred to as “crudediglycosidase”). This solution was applied to a hydrophobicchromatography (HiLoad 16/10 Phenyl Sepharose High Performance(Pharmacia)) to separate a fraction showing diglycosidase activity fromfractions showing β-glucosidase and β-xylosidase activities. Elution wasstarted at room temperature with 20 mM acetate buffer containing 30%saturated ammonium sulfate at a flow rate of 2 mL/min and elution wascarried out by linear gradient of 30 to 0%. A fraction showingdiglycosidase activity was eluted at 10 to 12.5% saturated ammoniumsulfate concentration. The diglycosidase fraction recovered wasconcentrated and a centrifuged supernatant was charged onto 10-DG columnto exchange the solution for 25 mM tris-hydrochloride buffer of pH 7.1.This liquid was applied to an isoelectric chromatography (Mono-P HR5/20(Pharmacia)) and elution was started at room temperature with polybuffer74 of pH 5.0 at 1 mL/min. The aimed diglycosidase activity was elutedfrom pH 6.2 to pH 6.3. Since a single band was obtained on an SDSelectrophoresis of the fraction, it was proved that diglycosidase(hereinafter, also referred to as “purified diglycosidase”) could bepurified.

EXAMPLE 2 Reactivity of Diglycosidase Toward Various IsoflavoneGlycosides

Diglycosidase was diluted with an acetate buffer of pH 4.0 to prepare a0.75 AU/mL enzyme solution. As references, a similar operation wasconducted using β-glucosidase (manufactured by Fluka) derived fromAspergillus niger, β-glucosidase (manufactured by Sigma) derived fromalmond, β-xylosidase derived from pectinase G (manufactured by AmanoEnzyme Inc.). Each of purified products of isoflavone glycosides(glycitin, acetylglycitin, malonylglycitin, daidzin, acetyldaidzin,malonyldaidzin, genistin, acetylgenistin, malonylgenistin, allmanufactured by Nacalai Tesque, Inc.) was dissolved in methanol toprepare each 2 mM substrate solution. The reaction was carried out bymixing 10 μL of a substrate solution, 200 μL of 20 mM acetate buffer (pH4.0), and 40 μL of each purified enzyme solution at a total liquidvolume of 250 μL. The reaction was carried out at 55° C. and isolationof an aglycon isoflavone from an isoflavone glycoside in the reactionmixture was detected by HLPC at 0, 1, 3, and 6 hours of the reaction.

HLPC Analysis

To the reaction mixture was added 700 μL of ethanol, followed bystirring and ultrasonication. After centrifugation at 15,000 rpm and 4°C. for 10 minutes, the supernatant was filtered through a filter andthen the filtrate was applied to HPLC.

The isoflavone glycoside and aglycon isoflavone contained in thefiltrate was separated and detected by a high performance liquidchromatography (HPLC, Shimadzu CLASS LC-10 system) using TOSOH TSK gelODS-80TM column (manufactured by Tosoh Corporation). The filtratecontaining an isoflavone glycoside and an aglycon isoflavone wasinjected into the column by means of an auto-injector (Shimadzu,SIL-10AXL) and elution was started with a solution containing 2% ofeluting solution A (acetonitrile) and 98% of eluting solution B (10%acetic acid solution), and after 5 minutes, continued by a linearconcentration gradient finishing with a solution of 50% of elutingsolution A and 50% of eluting solution B. Total flow rate was 0.8 mL/minand 12 kinds of isoflavone glycosides and aglycon isoflavones, i.e.,glycitin, daidzin, genistin, 6″-O-acetylglycitin, 6″-O-acetyldaidzin,6″-O-acetylgenistin, 6″-O-malonylglycitin, 6″-O-malonyldaidzin,6″-O-malonylgenistin, glycitein, daidzein, and genistein can beseparated. The absorbance at 260 nm was detected by a UV detector(Shimadzu, SPD-10AV). Using purified products (manufactured by NacalaiTesque, Inc.) of the above isoflavone glycosides and aglyconisoflavones, the isoflavone glycoside and aglycon isoflavone werequantitatively determined according to a calibration curve method. Bythe way, unless otherwise stated, the measuring conditions of HPLCherein mean those described in the example.

As a result, β-glucosidase derived from Aspergillus niger could act wellon 3 types of glucoside isoflavones of glycitin, daidzin, and genistin,but the reactivity on modified glycosides was very low. β-Glucosidasederived from a plant exhibits a low efficiency of decomposing glycosideisoflavones and no action was observed on modified isoflavones. On theother hand, diglycosidase could cleave all the isoflavone glycosides ata high efficiency. Of these, a high efficiency was observed towardacetylglucoside isoflavones. From the above results, diglycosidase wasfound to isolate aglycon isoflavones from isoflavone glycosides throughcleavage very efficiently (Table 1).

In addition, it was found that the efficiency of decomposing isoflavoneglucosides, especially genistin could be further enhanced by thecombined use of β-glucosidase in addition to the enzyme.

TABLE 1 Reaction time (h) 0 1 3 6 Purified enzyme Substrate glucosideaglycon glucoside aglycon glucoside aglycon glucoside aglyconDiglycosidase derived from glycitin 100 0 2.1 97.9 0.2 99.8 0.3 99.7 P.multicolor daidzin 99.1 0.9 29.9 70.1 18.2 81.8 11.5 88.5 genistin 100 0100 0 100 0 100 0 malonylglycitin 100 0 21.7 78.3 15.5 84.5 14 86malonyldaidzin 100 0 49.2 50.8 39.9 60.1 38.5 61.5 malonylgenistin 100 025.8 74.2 17.9 82.1 17.7 82.3 acetylglycitin 100 0 2 98 2.1 97.9 2.597.5 acetyldaidzin 100 0 10.4 89.6 3.8 96.2 3.8 96.2 acetylgenistin 1000 0.2 99.8 0.1 99.9 0.2 99.8 Diglycosidase derived from glycitin 100 099.5 0.5 100 0 100 0 A. fumigatus daidzin 99.1 0.9 92.9 7.1 85.3 14.775.2 24.8 genistin 100 0 100 0 100 0 100 0 malonylglycitin 100 0 99.60.4 99.2 0.8 98.6 1.4 malonyldaidzin 100 0 90 10 79.5 20.5 67.7 32.3malonylgenistin 100 0 97.6 2.4 94.8 5.2 91.8 8.2 acetylglycitin 100 0 946 86.5 13.5 77.3 22.7 acetyldaidzin 100 0 73.9 26.1 52.8 47.2 33.5 66.5acetylgenistin 100 0 87.4 12.6 77.1 22.9 64.8 35.2 β-Xylosidase derivedfrom glycitin 100 0 99.2 0.8 97.6 2.4 95.5 4.5 pectinase G daidzin 99.10.9 90.9 9.1 76 24 57.9 42.1 genistin 100 0 98.5 1.5 98.2 1.8 98.2 1.8malonylglycitin 100 0 100 0 100 0 99.6 0.4 malonyldaidzin 100 0 99.6 0.498.4 1.6 96.7 3.3 malonylgenistin 100 0 100 0 99.4 0.6 98 2acetylglycitin 100 0 100 0 100 0 100 0 acetyldaidzin 100 0 90.3 9.7 98.71.3 97.7 2.3 acetylgenistin 100 0 100 0 99.8 0.2 99.5 0.5 β-Glucosidasederived from glycitin 100 0 50.5 49.5 11.7 88.3 4.5 95.5 A. nigerdaidzin 100 0 0 100 0 100 0 100 genistin 100 0 0 100 0 100 0 100malonylglycitin 100 0 98.8 1.2 95.6 4.4 90.9 9.1 malonyldaidzin 100 095.5 4.5 90.7 9.3 84 16 malonylgenistin 100 0 96.9 3.1 93 7 87.9 12.1acetylglycitin 100 0 100 0 99 1 98.1 1.9 acetyldaidzin 100 0 95.6 4.493.6 6.4 91 9 acetylgenistin 100 0 98.6 1.4 96.7 3.3 93.8 6.2β-Glucosidase derived from glycitin 100 0 98.4 1.6 98.1 1.9 98.1 1.9almond daidzin 99.1 0.9 90.9 9.1 89.2 10.8 88.7 11.3 genistin 100 0 90.69.4 88.6 11.4 88.2 11.8 malonylglycitin 100 0 100 0 100 0 100 0malonyldaidzin 100 0 100 0 100 0 100 0 malonylgenistin 100 0 100 0 100 0100 0 acetylglycitin 100 0 100 0 100 0 100 0 acetyldaidzin 99.8 0.2 99.50.5 99.4 0.6 99.4 0.6 acetylgenistin 100 0 100 0 100 0 100 0

EXAMPLE 3 Influence of Free Glucose on Diglycosidase Activity

Purified diglycosidase was diluted with 20 mM acetate buffer of pH 4.0to prepare a 0.75 AU/mL enzyme solution.

With a glucose solution was mixed 10 μL of each 2 mM substrate solutiondescribed in Example 2, and 20 mM acetate buffer of pH 4.0 was addedthereto to be a liquid volume of 210 μL. Further, 40 μL of the enzymesolution was added and reaction was carried out at a final liquid volumeof 250 μL. Adjustment of the glucose solution to be added allows glucoseto exist in the reaction mixture in the range of 0 to 20%. The reactionwas carried out at 55° C. and the existence of isoflavone glycosides andaglycon isoflavones was detected by HLPC at 0, 0.5, 1, and 3 hours ofthe reaction.

When the results were compared assuming that isolated aglycon amount atthe glucose concentration of 0% and the reaction time of 0.5 hour is100% using each isoflavone glycoside as the substrate, the convertingefficiency of diglycosidase into aglycon isoflavone is hardly inhibitedby the increase of free glucose concentration. To the contrary, increaseof the converting efficiency was observed until 8% glucoseconcentration. Therefore, in the conversion into aglycon isoflavone bydiglycosidase, no inhibition by glucose was observed up to 20%concentration (Table 2).

Moreover, β-glucosidase used in the conventional converting method intoaglycon isoflavone is inhibited by glucose and large decrease of thereaction efficiency was observed, but diglycosidase of the invention wasfound to be hardly inhibited by glucose. Therefore, the amount, kind,and usage of phytogenic materials, which are starting materials forconverting into aglycon isoflavone, are restricted in the case of theconventional glycosidase, but there is no such restriction in the caseof diglycosidase of the invention and the efficiency of the conversioninto isoflavone aglycon is remarkably enhanced.

TABLE 2 Isolation of isoflavone aglycons by diglycosidase in presence ofglucose Glucose Concentration Isoflavone glycoside (%) glycitin daidzinacetylglycitin acetylgenistin acetyldaidzin malonylglycitinmalonylgenistin malonyldaidzin 0 100 100 100 100 100 100 100 100 2 96128.1 100.9 104.1 108.1 121.4 152 125.3 4 99.7 126 100.9 104.1 112.2127.6 164.7 132.1 8 100.1 129.1 100.9 104.1 115.8 128.6 163.5 125.4 2092.8 111.9 100.9 104.1 114.5 116.4 139.5 98.1

EXAMPLE 4 Examination of Temperature in the Conversion into IsoflavoneAglycons by Diglycosidase Using Soybean Materials

Into 400 μL of 20 mM acetate buffer of pH 4.0 was suspended 50 mg ofeach of various soybean materials (roasted soy flour (manufactured byFuji Shokuryo K.K.), soymilk (manufactured by Gitoh Shokuhin K.K.),defatted soybean (manufactured by Fuji Seiyu K.K.), concentrated soybeanprotein (manufactured by Fuji Seiyu K.K.)), whereby a substrate solutionwas prepared. Crude diglycosidase was diluted with 20 mM acetate bufferof pH 4.0 to be the glycosidase activity of 1.88 AU/mL. Fifty μL of theenzyme solution was mixed with the substrate solution and the whole wasreacted at 80, 65, 55, 45, 37, or 30° C. at the total volume of 500 μL.To the reaction mixture was added 700 μL of ethanol after 0, 1, 3, and 6hours of the reaction. After stirring and ultrasonication, the mixturewas subjected to centrifugal separation at 15,000 rpm and 4° C. for 10minutes. The supernatant was filtered through a filter and the existenceof isoflavone glycosides and aglycon isoflavones contained in thereaction mixture was detected by HLPC.

The decomposition efficiency from isoflavone glycosides into aglyconisoflavones by an enzyme preparation containing diglycosidase activitywas investigated during the treatment of 4 kinds of soybean materials(roasted soy flour, soymilk, concentrated soybean protein, defattedsoybean) with the enzyme, the isoflavone compounds being separated intothree groups. Namely, the conversion efficiency was analyzed upon threegroups of glycitin family (glycitin, malonylglycitin, acetylglycitin,glycitein), genistin family (genistin, malonylgenistin, acetylgenistin,genistein), and daidzin family (daidzin, malonyldaidzin, malonyldaidzin,acetyldaidzin, daidzein (Tables 3 to 14).

TABLE 3 Decomposition efficiency of glycitin family in roasted soy flourReaction Heat Reaction temperature treatment time Glycoside Aglycon (°C.) of enzyme (h) glycitin malonylglycitin acetylglycitin glycitein 30no 0 50.9 not detected 38.7 10.4 1 34.8 not detected 33.8 31.5 3 notdetected not detected 33.2 66.8 6 not detected not detected 19.1 80.9yes 6 51.4 not detected 37.5 11.1 45 no 0 51.3 not detected 38.3 10.4 112.4 not detected 27.4 60.2 3 12.4 not detected 20.3 67.4 6  5.9 notdetected  9.1 85.0 yes 6 49.8 not detected 36.8 13.4 55 no 0 50.5 notdetected 39.1 10.4 1 not detected not detected 30.1 69.9 3 not detectednot detected 12.1 87.9 6 not detected not detected  6.0 94.0 yes 6 51.7not detected 37.3 11.0 65 no 0 50.9 not detected 38.7 10.4 1 notdetected not detected 34.5 65.5 3 not detected not detected 31.0 69.0 6not detected not detected 29.0 71.0 yes 6 50.3 not detected 39.3 10.4 80no 0 51.3 not detected 38.3 10.4 1 19.2 not detected 40.1 40.8 3 20.5not detected 38.4 41.1 6 33.6 not detected 35.7 30.7 yes 6 50.6 notdetected 39.4  9.9 

TABLE 4 Decomposition efficiency of genistin family in roasted soy flourReaction Heat Reaction temperature treatment time Glycoside Aglycon (°C.) of enzyme (h) genistin malonylgenistin acetylgenistin genistein 30no 0 49.7 not detected 45.5  4.9 1 25.7 not detected 45.3 29.0 3 12.0not detected 43.8 44.2 6  5.5 not detected 40.6 53.8 yes 6 50.3 notdetected 43.8  5.8 45 no 0 50.2 not detected 45.3  4.4 1  3.7 notdetected 43.6 52.7 3  5.6 not detected 38.5 55.9 6  1.2 not detected31.7 67.1 yes 6 51.7 not detected 39.5  8.8 55 no 0 49.7 not detected45.7  4.6 1  1.2 not detected 42.5 56.2 3 not detected not detected 34.066.0 6 not detected not detected 27.3 72.7 yes 6 54.0 not detected 39.9 6.2 65 no 0 49.7 not detected 45.5  4.9 1 not detected not detected43.6 56.4 3 not detected not detected 40.5 59.5 6 not detected notdetected 38.8 61.2 yes 6 50.7 not detected 44.3  5.0 80 no 0 50.2 notdetected 45.3  4.4 1  7.8 not detected 48.5 43.6 3  9.1 not detected48.2 42.7 6 18.4 not detected 47.7 33.9 yes 6 52.2 not detected 43.4 4.3 

TABLE 5 Decomposition efficiency of daidzin family in roasted soy flourReaction Heat Reaction temperature treatment time Glycoside Aglycon (°C.) of enzyme (h) daidzin malonyldaidzin acetyldaidzin daidzein 35 no 048.6 not detected 47.8  3.6 1 11.7 not detected 46.9 41.4 3 not detectednot detected 45.3 54.7 6 not detected not detected 39.6 60.4 yes 6 50.3not detected 43.8  5.8 45 no 0 48.6 not detected 47.7  3.7 1  3.6 notdetected 42.9 53.5 3  2.7 not detected 37.9 59.4 6  5.4 not detected28.5 66.2 yes 6 49.4 not detected 42.5  8.1 55 no 0 48.8 not detected47.5  3.7 1 not detected not detected 42.8 57.2 3 not detected notdetected 32.5 67.5 6 not detected not detected 26.7 73.3 yes 6 51.1 notdetected 43.6  5.3 65 no 0 48.6 not detected 47.8  3.6 1 not detectednot detected 43.7 56.3 3 not detected not detected 40.2 59.8 6 notdetected not detected 37.8 62.2 yes 6 49.7 not detected 46.1  4.2 80 no0 48.6 not detected 47.7  3.7 1  3.2 not detected 49.1 47.7 3  4.4 notdetected 47.9 47.7 6 12.5 not detected 46.7 40.8 yes 6 51.3 not detected45.0  3.8 

TABLE 6 Decomposition efficiency of glycitin family in soymilk ReactionHeat Reaction temperature treatment time Glycoside Aglycon (° C.) ofenzyme (h) glycitin malonylglycitin acetylglycitin glycitein 30 no 043.9 48.0 not detected 8.1 1 not detected 51.0 not detected 49.0 3 notdetected 39.6 not detected 60.4 6 not detected 28.4 not detected 71.6yes 6 44.5 47.7 not detected 7.9 45 no 0 42.6 49.1 not detected 8.2 1not detected 46.2 not detected 53.8 3 not detected 29.9 not detected70.1 6 not detected 15.3 not detected 84.7 yes 6 45.4 47.2 not detected7.4 55 no 0 47.8 44.6 not detected 7.6 1 not detected 39.1 not detected60.9 3 not detected 27.7 not detected 72.3 6 not detected 16.5 notdetected 83.5 yes 6 53.5 39.9 not detected 6.7 65 no 0 43.9 48.0 notdetected 8.1 1 not detected 42.4 not detected 57.6 3 not detected 34.3not detected 65.7 6 not detected 25.5 not detected 74.5 yes 6 56.8 36.0not detected 7.2 80 no 0 42.6 49.1 not detected 8.2 1 10.7 43.6 notdetected 45.7 3 22.5 32.6 not detected 44.9 6 44.2 21.3 not detected34.4 yes 6 71.4 19.3 not detected 9.3 

TABLE 7 Decomposition efficiency of genistin family in soymilk ReactionHeat Reaction temperature treatment time Glycoside Aglycon (° C.) ofenzyme (h) genistin malonylgenistin acetylgenistin genistein 30 no 030.7 58.5 0.9 10.0 1 not detected 56.5 not detected 43.5 3 not detected47.9 not detected 52.1 6 not detected 36.1 not detected 63.9 yes 6 31.257.7 0.9 10.1 45 no 0 31.0 57.8 0.9 10.3 1 not detected 49.4 notdetected 50.6 3 not detected 34.7 not detected 65.3 6 not detected 20.7not detected 79.3 yes 6 34.4 57.2 0.6  7.9 55 no 0 31.6 59.1 0.6  8.7 1not detected 46.9 not detected 53.1 3 not detected 32.5 not detected67.5 6 not detected 20.4 not detected 79.6 yes 6 37.4 53.3 0.7  8.6 65no 0 30.7 58.5 0.9 10.0 1 not detected 48.9 not detected 51.1 3 notdetected 40.6 not detected 59.4 6 not detected 31.8 not detected 68.2yes 6 44.4 44.6 1.1  9.9 80 no 0 31.0 57.8 0.9 10.3 1 10.6 50.4 0.9 38.03 24.3 37.7 1.0 37.0 6 43.9 24.1 2.3 29.6 yes 6 64.5 22.9 2.5 10.1 

TABLE 8 Decomposition efficiency of daidzin family in soymilk ReactionHeat Reaction temperature treatment time Glycoside Aglycon (° C.) ofenzyme (h) daidzin malonyldaidzin acetyldaidzin daidzein 30 no 0 34.156.8 not detected 9.1 1 not detected 58.2 not detected 41.8 3 notdetected 52.8 not detected 47.2 6 not detected 46.2 not detected 53.8yes 6 34.8 56.1 not detected 9.1 45 no 0 33.9 57.2 not detected 8.9 1 6.8 50.2 not detected 43.0 3  6.9 41.1 not detected 52.0 6  6.9 30.2not detected 62.8 yes 6 36.0 55.8 not detected 8.2 55 no 0 34.2 57.3 notdetected 8.6 1 not detected 50.9 not detected 49.1 3 not detected 41.1not detected 58.9 6 not detected 31.1 not detected 68.9 yes 6 40.7 50.8not detected 8.6 65 no 0 34.1 56.8 not detected 9.1 1 not detected 51.7not detected 48.3 3 not detected 43.9 not detected 56.1 6 not detected35.3 not detected 64.7 yes 6 48.7 42.4 not detected 8.8 80 no 0 33.957.2 not detected 8.9 1  8.9 50.8 not detected 40.3 3 23.9 36.4 notdetected 39.7 6 42.9 23.0 not detected 34.1 yes 6 69.5 21.8 not detected8.8 

TABLE 9 Decomposition efficiency of glycitin family in concentratedsoybean protein Reaction Heat Reaction temperature treatment timeGlycoside Aglycon (° C.) of enzyme (h) glycitin malonylglycitinacetylglycitin glycitein 30 no 0 52.2 0.4 36.0 11.4 1 47.1 0.5 35.0 17.53 39.1 0.5 33.4 26.9 6 28.4 0.5 30.8 40.3 yes 6 52.8 0.4 35.4 11.3 45 no0 52.3 0.4 35.9 11.4 1 30.2 0.6 34.1 35.2 3 18.8 0.6 31.0 49.6 6  7.50.6 27.1 64.9 yes 6 53.2 0.5 35.0 11.3 55 no 0 52.3 0.4 36.0 11.2 1 14.20.5 33.9 51.4 3 not detected 0.5 29.8 69.7 6 not detected 0.5 26.8 72.7yes 6 52.9 0.4 35.3 11.3 65 no 0 52.2 0.4 36.0 11.4 1  5.2 0.5 35.7 58.63 not detected 0.5 35.5 64.0 6 not detected 0.4 35.8 63.8 yes 6 52.5 0.435.7 11.4 80 no 0 52.3 0.4 35.9 11.4 1 39.7 0.4 36.0 23.8 3 40.5 0.435.7 23.4 6 41.7 0.3 35.2 22.8 yes 6 53.6 0.3 34.8 11.4 

TABLE 10 Decomposition efficiency of genistin family in concentratedsoybean protein Reaction Heat Reaction temperature treatment timeGlycoside Aglycon (° C.) of enzyme (h) genistin malonylgenistinacetylgenistin genistein 30 no 0 49.8 not detected 43.1 7.1 1 41.1 notdetected 43.2 15.7 3 30.3 not detected 43.0 26.7 6 19.7 not detected42.5 37.8 yes 6 51.8 not detected 41.5 6.7 45 no 0 49.7 not detected43.0 7.2 1 22.2 not detected 42.8 35.0 3 15.6 not detected 41.0 43.4 6 5.3 not detected 39.6 55.1 yes 6 54.5 not detected 38.9 6.5 55 no 050.1 not detected 42.9 7.0 1  7.0 not detected 43.1 49.8 3  0.8 notdetected 41.1 58.1 6 not detected not detected 39.0 61.0 yes 6 52.3 notdetected 41.1 6.6 65 no 0 49.8 not detected 43.1 7.1 1 not detected notdetected 45.2 54.8 3 not detected not detected 44.0 56.0 6 not detectednot detected 43.4 56.6 yes 6 50.8 not detected 42.3 6.9 80 no 0 49.7 notdetected 43.0 7.2 1 29.6 not detected 44.0 26.4 3 30.3 not detected 43.626.1 6 32.3 not detected 43.4 24.3 yes 6 51.9 not detected 41.4 6.7 

TABLE 11 Decomposition efficiency of daidzin family in concentratedsoybean protein Reaction Heat Reaction temperature treatment timeGlycoside Aglycon (° C.) of enzyme (h) daidzin malonyldaidzinacetyldaidzin daidzein 30 no 0 52.3 not detected 44.6 3.0 1 29.5 notdetected 44.4 26.2 3 13.4 not detected 43.9 42.7 6  4.0 not detected43.5 52.5 yes 6 54.5 not detected 42.3 3.2 45 no 0 52.4 not detected44.5 3.1 1 10.6 not detected 44.1 45.3 3  9.6 not detected 41.5 48.8 6 1.7 not detected 42.9 55.5 yes 6 94.3 not detected  0.0 5.7 55 no 052.5 not detected 44.5 3.0 1  1.0 not detected 44.0 55.0 3 not detectednot detected 40.6 59.4 6 not detected not detected 37.7 62.3 yes 6 54.6not detected 42.3 3.2 65 no 0 52.3 not detected 44.6 3.0 1  1.8 notdetected 44.3 53.9 3  1.7 not detected 42.9 55.4 6  1.7 not detected41.6 56.7 yes 6 53.2 not detected 43.6 3.2 80 no 0 52.4 not detected44.5 3.1 1 22.6 not detected 45.5 31.9 3 23.3 not detected 44.7 32.0 625.5 not detected 43.8 30.7 yes 6 54.5 not detected 42.3 3.2 

TABLE 12 Decomposition efficiency of glycitin family in defatted soybeanReaction Heat Reaction temperature treatment time Glycoside Aglycon (°C.) of enzyme (h) glycitin malonylglycitin acetylglycitin glycitein 30no 0 not detected 61.3 not detected 38.7 1 not detected 48.3 notdetected 51.7 3 not detected 44.6 not detected 55.4 6 not detected 45.6not detected 54.4 yes 6 not detected 45.1 not detected 54.9 45 no 0 notdetected 65.2 not detected 34.8 1 not detected 44.3 not detected 55.7 3not detected 44.8 not detected 55.2 6 not detected 41.0 not detected59.0 yes 6 not detected 44.2 not detected 55.8 55 no 0 not detected 61.3not detected 38.7 1 not detected 44.8 not detected 55.2 3 not detected42.6 not detected 57.4 6 not detected 42.7 not detected 57.3 yes 6 notdetected 45.5 not detected 54.5 65 no 0 not detected 65.2 not detected34.8 1 not detected 45.3 not detected 54.7 3 not detected 42.1 notdetected 57.9 6 not detected 40.0 not detected 60.0 yes 6 not detected48.8 not detected 51.2 80 no 0 not detected 65.2 not detected 34.8 1 notdetected 46.1 not detected 53.9 3 not detected 45.0 not detected 55.0 6not detected 40.3 not detected 59.7 yes 6 not detected 48.1 not detected51.9 

TABLE 13 Decomposition efficiency of genistin family in defatted soybeanReaction Heat Reaction temperature treatment time Glycoside Aglycon (°C.) of enzyme (h) genistin malonylgenistin acetylgenistin genistein 30no 0 38.0 47.0 1.7 13.3 1 not detected 51.3 1.3 47.4 3 not detected 49.10.7 50.2 6  7.6 45.6 0.1 46.6 yes 6 not detected 49.5 0.4 50.2 45 no 037.0 47.0 1.5 14.5 1  0.9 49.0 0.8 49.2 3  1.0 46.8 0.3 51.8 6  1.0 44.20.2 54.6 yes 6  1.3 48.1 0.2 50.4 55 no 0 38.0 47.0 1.7 13.3 1  0.0 48.80.7 50.5 3  0.0 46.1 0.2 53.7 6  0.0 43.4 0.1 56.5 yes 6  0.0 47.8 0.451.9 65 no 0 37.0 47.0 1.5 14.5 1  0.0 48.9 1.2 49.9 3  1.8 45.2 1.251.8 6  3.3 41.1 1.1 54.5 yes 6 28.2 39.7 1.3 30.7 80 no 0 37.0 47.0 1.514.5 1  9.6 43.8 1.8 44.9 3 22.1 32.5 2.1 43.3 6 35.3 20.9 2.5 41.3 yes6 57.8 20.1 2.4 19.6 

TABLE 14 Decomposition efficiency of daidzin family in defatted soybeanReaction Heat Reaction temperature treatment time Glycoside Aglycon (°C.) of enzyme (h) daidzin malonyldaidzin acetyldaidzin daidzein 30 no 042.2 43.3 not detected 14.5 1 not detected 46.6 not detected 53.4 3 notdetected 45.2 not detected 54.8 6 not detected 45.1 not detected 54.9yes 6 not detected 45.4 not detected 54.6 45 no 0 40.5 43.3 not detected16.2 1 not detected 45.5 not detected 54.5 3 not detected 43.7 notdetected 56.3 6 not detected 41.7 not detected 58.3 yes 6 not detected44.3 not detected 55.7 55 no 0 42.2 43.3 not detected 14.5 1 notdetected 45.0 not detected 55.0 3 not detected 42.7 not detected 57.3 6not detected 40.3 not detected 59.7 yes 6 not detected 44.1 not detected55.9 65 no 0 40.5 43.3 not detected 16.2 1 not detected 44.7 notdetected 55.3 3 not detected 41.6 not detected 58.4 6 not detected 37.3not detected 62.7 yes 6 32.8 35.2 not detected 32.0 80 no 0 40.5 43.3not detected 16.2 1  7.8 39.8 not detected 52.4 3 19.2 28.8 not detected52.0 6 30.4 17.8 not detected 51.7 yes 6 60.9 17.4 not detected 21.7

All three groups of isoflavone glucosides were decomposed at all thetemperature ranges tested, and promptly at 37 to 65° C., particularly55° C. Furthermore, in defatted soybean, endogenous β-glucosidase alsoparticipates in the decomposition. It is revealed that the decompositionof modified glucoside glycosides, most of which is considered to occurby the action of diglycosidase, easily occurs at a temperature of 37 to55° C. Among three groups of aglycon isoflavones, maximum isolation ofaglycons was observed in the reaction at 55° C. for 6 hours. In the caseof soy flour, glycitein was about 94%, genistein about 74%, and daidzeinabout 73%. In the case of soymilk, glycitein was about 84%, genisteinabout 80%, and daidzein about 70%. In the case of concentrated soybeanprotein, glycitein was about 73%, genistein about 61%, and daidzeinabout 62%. In the case of defatted soybean, glycitein was about 57%,genistein about 57%, and daidzein about 60%.

From these results, it was revealed that the decomposition of modifiedglucoside glycosides by diglycosidase efficiently occurred at atemperature of 37 to 65° C., particularly around 55° C.

EXAMPLE 5 Examination of pH in the Conversion into Isoflavone Aglyconsby Diglycosidase Using Soybean Materials

A substrate solution of 450 μL was prepared by suspending 50 mg of eachsoybean material (roasted soy flour (manufactured by Fuji ShokuryoK.K.), soymilk (manufactured by Gitoh Shokuhin K.K.), defatted soybean(manufactured by Fuji Seiyu K.K.), concentrated soybean protein(manufactured by Fuji Seiyu K.K.)), and adjusting the pH to 2 to 11 withhydrochloric acid or sodium hydroxide. Each enzyme solution of pH 2–11wherein diglycosidase activity of crude diglycosidase was adjusted to1.88 AU/mL was added thereto in an amount of 50 μL and the whole wasreacted at 55° C. at the total volume of 500 μL. To the reaction mixturewas added 700 μL of ethanol after 0, 1, 3, and 6 hours of the reaction.After stirring and ultrasonication, the mixture was subjected tocentrifugal separation at 15,000 rpm and 4° C. for 10 minutes. Thesupernatant was filtered through a filter and the existence ofisoflavone glycosides and aglycon isoflavones contained in the reactionmixture was detected by HLPC (Tables 15 to 26).

TABLE 15 Decomposition efficiency of glycitin family in roasted soyflour Heat Reaction treatment time Glycoside Aglycon Reaction pH ofenzyme (h) glycitin malonylglycitin acetylglycitin glycitein pH2 no 056.8 not detected 35.3 7.9 1 56.6 not detected 34.0 9.4 3 56.3 notdetected 34.2 9.5 6 54.4 not detected 35.5 10.1 yes 6 54.4 not detected35.2 10.4 pH3 no 0 56.8 not detected 35.3 7.9 1 not detected notdetected 38.7 61.3 3 not detected not detected 30.8 69.2 6 not detectednot detected 31.7 68.3 yes 6 55.1 not detected 35.6 9.3 pH4 no 0 56.6not detected 34.8 8.6 1 not detected not detected 18.8 81.2 3 notdetected not detected  8.2 91.8 6 not detected not detected not detected100.0 yes 6 55.1 not detected 35.9 9.0 pH5 no 0 56.6 not detected 34.88.6 1 not detected not detected 22.9 77.1 3 not detected not detected 6.5 93.5 6 not detected not detected not detected 100.0 yes 6 55.4 notdetected 35.2 9.4 pH6.5 no 0 55.9 not detected 35.4 8.8 1 not detectednot detected 35.0 65.0 3 not detected not detected 19.7 80.3 6 notdetected not detected 11.9 88.1 yes 6 54.8 not detected 35.4 9.8 pH8.5no 0 59.4 not detected 31.0 9.6 1 39.4 not detected 32.4 28.2 3 29.3 notdetected 30.6 40.1 6 22.4 not detected 30.2 47.4 yes 6 59.2 not detected31.7 9.1 

TABLE 16 Decomposition efficiency of genistin family in roasted soyflour Heat Reaction treatment time Glycoside Aglycon Reaction pH ofenzyme (h) genistin malonylgenistin acetylgenistin genistein pH2 no 050.0 not detected 45.2 4.8 1 49.7 not detected 45.0 5.3 3 50.7 notdetected 43.1 6.2 6 52.7 not detected 40.7 6.5 yes 6 52.8 not detected41.2 6.0 pH3 no 0 50.0 not detected 45.2 4.8 1  3.4 not detected 45.251.4 3 not detected not detected 41.9 58.1 6 not detected not detected43.5 56.5 yes 6 49.9 not detected 45.1 5.0 pH4 no 0 49.9 not detected45.4 4.7 1 not detected not detected 36.3 63.7 3 not detected notdetected 23.4 76.6 6 not detected not detected 13.4 86.6 yes 6 50.2 notdetected 45.4 4.4 pH5 no 0 49.9 not detected 45.4 4.7 1 not detected notdetected 39.9 60.1 3 not detected not detected 26.9 73.1 6 not detectednot detected 17.6 82.4 yes 6 52.6 not detected 42.0 5.3 pH6.5 no 0 49.4not detected 45.8 4.8 1 not detected not detected 45.3 54.7 3 notdetected not detected 38.8 61.2 6 not detected not detected 34.0 66.0yes 6 54.8 not detected 39.2 6.0 pH8.5 no 0 55.8 not detected 39.5 4.6 128.7 not detected 39.5 31.8 3 19.2 not detected 39.2 41.6 6 14.5 notdetected 37.3 48.2 yes 6 59.5 not detected 35.2 5.3 

TABLE 17 Decomposition efficiency of daidzin family in roasted soy flourHeat Reaction treatment time Glycoside Aglycon Reaction pH of enzyme (h)daidzin malonyldaidzin acetyldaidzin daidzein pH2 no 0 49.1 not detected47.4 3.5 1 47.6 not detected 46.9 5.4 3 50.3 not detected 45.0 4.8 653.1 not detected 42.4 4.5 yes 6 53.8 not detected 42.4 3.8 pH3 no 049.1 not detected 47.4 3.5 1 not detected not detected 46.0 54.0 3 notdetected not detected 39.6 60.4 6 not detected not detected 40.7 59.3yes 6 49.0 not detected 47.4 3.7 pH4 no 0 48.7 not detected 47.7 3.5 1not detected not detected 34.4 65.6 3 not detected not detected 20.579.5 6 not detected not detected 11.1 88.9 yes 6 48.6 not detected 47.93.5 pH5 no 0 48.7 not detected 47.7 3.5 1 not detected not detected 38.561.5 3 not detected not detected 24.7 75.3 6 not detected not detected15.2 84.8 yes 6 50.6 not detected 45.0 4.4 pH6.5 no 0 48.4 not detected48.2 3.4 1 not detected not detected 45.4 54.6 3 not detected notdetected 39.2 60.8 6 not detected not detected 34.4 65.6 yes 6 51.9 notdetected 43.4 4.7 pH8.5 no 0 54.1 not detected 42.1 3.8 1 20.4 notdetected 42.7 36.9 3 13.3 not detected 40.3 46.4 6 10.2 not detected38.0 51.8 yes 6 58.8 not detected 37.3 3.9 

TABLE 18 Decomposition efficiency of glycitin family in soymilk HeatReaction treatment time Glycoside Aglycon Reaction pH of enzyme (h)glycitin malonylglycitin acetylglycitin glycitein pH2.3 no 0 50.2 42.6not detected 7.2 1 not detected 51.3 not detected 48.7 3 not detected47.7 not detected 52.3 6 not detected 46.5 not detected 53.5 yes 6 51.741.3 not detected 6.9 pH3.5 no 0 50.2 42.6 not detected 7.2 1 notdetected 21.9 not detected 78.1 3 not detected not detected not detected100.0 6 not detected not detected not detected 100.0 yes 6 54.2 38.2 notdetected 7.6 pH4.8 no 0 50.7 43.2 not detected 6.2 1 not detected 37.0not detected 63.0 3 not detected 21.1 not detected 78.9 6 not detected 8.3 not detected 91.7 yes 6 54.3 39.2 not detected 6.5 pH6.2 no 0 50.743.2 not detected 6.2 1 not detected 46.4 not detected 53.6 3 notdetected 34.5 not detected 65.5 6 not detected 29.6 not detected 70.4yes 6 54.2 37.6 not detected 8.2 pH7.2 no 0 50.0 43.9 not detected 6.1 1not detected 48.8 not detected 51.2 3 not detected 46.2 not detected53.8 6 not detected 41.6 not detected 58.4 yes 6 56.9 37.5 not detected5.6 pH11.6 no 0 54.3 39.4 not detected 6.2 1 not detected 50.4 notdetected 49.6 3 not detected 46.5 not detected 53.5 6 not detected 44.0not detected 56.0 yes 6 59.7 34.4 not detected 5.8 

TABLE 19 Decomposition efficiency of genistin family in soymilk HeatReaction treatment time Glycoside Aglycon Reaction pH of enzyme (h)genistin malonylgenistin acetylgenistin genistein pH2.3 no 0 31.5 58.20.6 9.8 1 not detected 57.5 0.7 41.8 3 not detected 54.1 0.8 45.1 6 notdetected 52.1 0.6 47.3 yes 6 35.5 54.3 0.6 9.6 pH3.5 no 0 31.5 58.2 0.69.8 1 not detected 27.9 not detected 72.1 3 not detected 7.5 notdetected 92.5 6 not detected 2.7 not detected 97.3 yes 6 37.0 52.7 0.59.8 pH4.8 no 0 31.7 58.6 0.5 9.1 1 not detected 41.0 not detected 59.0 3not detected 23.9 not detected 76.1 6 not detected 11.3 not detected88.7 yes 6 37.8 51.9 0.6 9.7 pH6.2 no 0 31.7 58.6 0.5 9.1 1 not detected52.6 not detected 47.4 3 not detected 41.2 not detected 58.8 6 notdetected 35.8 not detected 64.2 yes 6 38.3 51.5 0.6 9.5 pH7.2 no 0 31.257.0 0.7 11.1 1 not detected 56.9 not detected 43.1 3 not detected 51.5not detected 48.5 6 not detected 46.4 not detected 53.6 yes 6 38.9 51.10.7 9.3 pH11.6 no 0 34.8 54.2 0.5 10.5 1 not detected 57.7 not detected42.3 3 not detected 52.6 not detected 47.4 6 not detected 49.5 notdetected 50.5 yes 6 41.3 47.7 0.9 10.1 

TABLE 20 Decomposition efficiency of daidzin family in soymilk HeatReaction treatment time Glycoside Aglycon Reaction pH of enzyme (h)daidzin malonyldaidzin acetyldaidzin daidzein pH2.3 no 0 34.4 55.1 notdetected 10.5 1 not detected 54.7 not detected 45.3 3 not detected 51.1not detected 48.9 6 not detected 48.5 not detected 51.5 yes 6 38.8 51.0not detected 10.2 pH3.5 no 0 34.4 55.1 not detected 10.5 1 not detected35.6 not detected 64.4 3 not detected 15.1 not detected 84.9 6 notdetected  7.4 not detected 92.6 yes 6 40.4 49.1 not detected 10.6 pH4.8no 0 34.9 56.2 not detected 9.0 1 not detected 47.4 not detected 52.6 3not detected 33.9 not detected 66.1 6 not detected 20.3 not detected79.7 yes 6 41.2 49.7 not detected 9.1 pH6.2 no 0 34.9 56.2 not detected9.0 1 not detected 54.1 not detected 45.9 3 not detected 46.4 notdetected 53.6 6 not detected 41.9 not detected 58.1 yes 6 40.6 49.7 notdetected 9.7 pH7.2 no 0 34.9 56.1 not detected 9.0 1 not detected 56.4not detected 43.6 3 not detected 52.4 not detected 47.6 6 not detected48.2 not detected 51.8 yes 6 41.5 48.3 not detected 10.2 pH11.6 no 038.8 52.5 not detected 8.7 1 not detected 56.4 not detected 43.6 3 notdetected 50.4 not detected 49.6 6 not detected 47.7 not detected 52.3yes 6 44.6 46.1 not detected 9.3 

TABLE 21 Decomposition efficiency of glycitin family in concentratedsoybean protein Heat treatment Reaction Reaction of time GlycosideAglycon pH enzyme (h) glycitin malonylglycitin acetylglycitin glyciteinpH1.6 no 0 52.3 0.4 35.9 11.3 1 52.9 0.4 35.1 11.6 3 54.3 0.4 33.5 11.76 56.1 0.4 31.8 11.7 yes 6 57.9 0.4 30.9 10.8 pH2.7 no 0 52.3 0.4 35.911.3 1 38.0 0.5 36.6 25.0 3 37.2 0.5 36.5 25.7 6 33.7 0.5 36.6 29.2 yes6 52.7 0.4 35.4 11.5 pH3.7 no 0 52.4 0.4 36.1 11.1 1 not detected 0.532.9 66.7 3 not detected 0.4 26.1 73.5 6 not detected 0.4 22.1 77.5 yes6 52.4 0.4 35.9 11.3 pH5.1 no 0 52.4 0.4 36.1 11.1 1 4.1 0.6 33.2 62.1 3not detected 0.6 26.0 73.4 6 not detected 0.6 21.0 78.5 yes 6 52.3 0.435.8 11.5 pH6.6 no 0 52.2 0.4 36.1 11.3 1 22.6 0.4 34.8 42.2 3 6.9 0.432.4 60.3 6 2.0 0.4 29.5 68.1 yes 6 53.3 0.4 35.2 11.1 pH8.6 no 0 54.90.4 33.0 11.7 1 51.6 0.4 32.6 15.4 3 45.9 0.4 32.6 21.0 6 36.7 0.4 32.030.9 yes 6 56.8 0.3 31.6 11.3 

TABLE 22 Decomposition efficiency of genistin family in concentratedsoybean protein Heat treatment Reaction Reaction of time GlycosideAglycon pH enzyme (h) genistin malonylgenistin acetylgenistin genisteinpH1.6 no 0 49.7 not detected 42.5 7.8 1 50.2 not detected 42.2 7.6 351.6 not detected 40.6 7.8 6 53.3 not detected 38.9 7.9 yes 6 54.4 notdetected 37.7 7.9 pH2.7 no 0 49.7 not detected 42.5 7.8 1 23.1 notdetected 44.3 32.6 3 22.1 not detected 43.7 34.2 6 17.5 not detected44.0 38.4 yes 6 50.5 not detected 42.5 7.0 pH3.7 no 0 50.7 not detected43.1 6.2 1 not detected not detected 43.5 56.5 3 not detected notdetected 36.9 63.1 6 not detected not detected 30.7 69.3 yes 6 50.6 notdetected 43.0 6.4 pH5.1 no 0 50.7 not detected 43.1 6.2 1 not detectednot detected 45.2 54.8 3 not detected not detected 40.2 59.8 6 notdetected not detected 35.6 64.4 yes 6 51.4 not detected 42.4 6.2 pH6.6no 0 50.4 not detected 43.0 6.5 1 13.9 not detected 43.6 42.4 3 3.8 notdetected 42.8 53.3 6 1.7 not detected 41.0 57.2 yes 6 53.2 not detected40.6 6.2 pH8.6 no 0 53.8 not detected 39.7 6.5 1 50.4 not detected 38.810.8 3 45.1 not detected 39.0 15.8 6 36.3 not detected 38.5 25.2 yes 656.2 not detected 37.6 6.2 

TABLE 23 Decomposition efficiency of daidzin family in concentratedsoybean protein Heat treatment Reaction Reaction of time GlycosideAglycon pH enzyme (h) daidzin malonyldaidzin acetyldaidzin daidzeinpH1.6 no 0 52.5 not detected 44.5 3.0 1 53.0 not detected 43.7 3.3 355.0 not detected 41.7 3.3 6 57.2 not detected 39.4 3.4 yes 6 59.0 notdetected 38.1 2.9 pH2.7 no 0 52.5 not detected 44.5 3.0 1 12.9 notdetected 46.4 40.7 3 12.5 not detected 46.0 41.6 6 9.3 not detected 45.845.0 yes 6 53.0 not detected 43.9 3.2 pH3.7 no 0 52.5 not detected 44.63.0 1 not detected not detected 41.6 58.4 3 not detected not detected33.0 67.0 6 not detected not detected 25.2 74.8 yes 6 52.5 not detected44.4 3.1 pH5.1 no 0 52.5 not detected 44.6 3.0 1 not detected notdetected 43.4 56.6 3 not detected not detected 37.0 63.0 6 not detectednot detected 31.2 68.8 yes 6 53.4 not detected 43.4 3.2 pH6.6 no 0 52.1not detected 44.8 3.0 1 5.0 not detected 43.9 51.0 3 not detected notdetected 42.9 57.1 6 not detected not detected 41.2 58.8 yes 6 55.1 notdetected 41.8 3.2 pH8.6 no 0 56.1 not detected 40.7 3.2 1 50.8 notdetected 39.2 9.9 3 45.0 not detected 39.0 15.9 6 34.5 not detected 38.127.4 yes 6 59.1 not detected 37.7 3.2 

TABLE 24 Decomposition efficiency of glycitin family in defatted soybeanHeat treatment Reaction Reaction of time Glycoside Aglycon pH enzyme (h)glycitin malonylglycitin acetylglycitin glycitein pH2.6 no 0 notdetected 100.0 not detected not detected 1 not detected 100.0 notdetected not detected 3 not detected 100.0 not detected not detected 6not detected 100.0 not detected not detected yes 6 not detected 100.0not detected not detected pH3.4 no 0 not detected 100.0 not detected notdetected 1 not detected 100.0 not detected not detected 3 not detected100.0 not detected not detected 6 not detected 100.0 not detected notdetected yes 6 not detected 100.0 not detected not detected pH4.8 no 0not detected 64.0 not detected 36.0 1 not detected 40.1 not detected59.9 3 not detected 37.5 not detected 62.5 6 not detected 31.9 notdetected 68.1 yes 6 not detected 56.2 not detected 43.8 pH5.4 no 0 notdetected 64.0 not detected 36.0 1 not detected 45.1 not detected 54.9 3not detected 100.0 not detected not detected 6 not detected 32.3 notdetected 67.7 yes 6 not detected 39.3 not detected 60.7 pH6.6 no 0 notdetected 39.6 not detected 60.4 1 not detected 52.9 not detected 47.1 3not detected 53.6 not detected 46.4 6 not detected 58.1 not detected41.9 yes 6 not detected 50.3 not detected 49.7 pH7.8 no 0 not detected66.8 not detected 33.2 1 not detected 57.5 not detected 42.5 3 notdetected 53.2 not detected 46.8 6 not detected 49.1 not detected 50.9yes 6 not detected 55.8 not detected 44.2 

TABLE 25 Decomposition efficiency of genistin family in defatted soybeanHeat treatment Reaction Reaction of time Glycoside Aglycon pH enzyme (h)genistin malonylgenistin acetylgenistin genistein pH2.6 no 0 42.0 43.81.8 12.4 1 36.3 44.4 1.7 17.7 3 20.3 45.4 1.7 32.7 6 20.1 43.5 1.9 34.6yes 6 44.3 42.0 1.6 12.1 pH3.4 no 0 42.0 43.8 1.8 12.4 1 not detected41.6 1.5 56.9 3 not detected 36.4 1.1 62.5 6 not detected 30.4 0.8 68.8yes 6 44.3 39.8 1.8 14.1 pH4.8 no 0 40.5 43.2 1.8 14.5 1 not detected42.8 1.0 56.1 3 not detected 37.5 0.6 61.9 6 not detected 31.1 0.4 68.5yes 6 31.8 41.7 1.3 25.2 pH5.4 no 0 40.5 43.2 1.8 14.5 1 not detected48.0 0.5 51.5 3 not detected 44.6 not detected 55.4 6 not detected 39.7not detected 60.3 yes 6 6.2 45.8 not detected 48.0 pH6.6 no 0 39.2 47.11.7 12.0 1 not detected 50.1 0.6 49.3 3 not detected 48.3 0.3 51.4 6 notdetected 46.1 0.2 53.7 yes 6 7.7 45.7 0.2 46.4 pH7.8 no 0 40.4 45.8 1.612.2 1 15.7 46.6 1.0 36.7 3 12.3 44.4 0.8 42.5 6 11.4 42.2 0.7 45.7 yes6 37.5 39.9 0.6 22.0 

TABLE 26 Decomposition efficiency of daidzin family in defatted soybeanHeat treatment Reaction Reaction of time Glycoside Aglycon pH enzyme (h)daidzin malonyldaidzin acetyldaidzin daidzein pH2.6 no 0 45.8 41.0 notdetected 13.2 1 37.5 41.2 not detected 21.3 3 17.3 41.8 not detected40.9 6 16.9 40.2 not detected 42.9 yes 6 48.0 38.9 not detected 13.0pH3.4 no 0 45.8 41.0 not detected 13.2 1 not detected 39.4 not detected60.6 3 not detected 34.5 not detected 65.5 6 not detected 29.0 notdetected 71.0 yes 6 47.8 37.1 not detected 15.1 pH4.8 no 0 43.5 40.7 notdetected 15.7 1 not detected 40.5 not detected 59.5 3 not detected 36.7not detected 63.3 6 not detected 30.5 not detected 69.5 yes 6 37.1 38.4not detected 24.5 pH5.4 no 0 43.5 40.7 not detected 15.7 1 not detected44.2 not detected 55.8 3 not detected 41.5 not detected 58.5 6 notdetected 36.9 not detected 63.1 yes 6 not detected 44.9 not detected55.1 pH6.6 no 0 43.6 43.1 not detected 13.2 1 not detected 45.9 notdetected 54.1 3 not detected 44.0 not detected 56.0 6 not detected 41.8not detected 58.2 yes 6 9.3 41.2 not detected 49.5 pH7.8 no 0 44.7 42.0not detected 13.3 1 13.3 43.6 not detected 43.1 3 10.1 41.4 not detected48.4 6 9.4 38.7 not detected 51.9 yes 6 41.6 36.7 not detected 21.7

From these results, maximum pH was found to be in the range of 3.5 to 5.Specifically, in the case of roasted soy flour, after the reaction at pH4 for 6 hours, the existing ratio of aglycon of each isoflavone familywas as follows: glycitein 100%, genistein 87%, and daidzein 89%. In thecase of soymilk, almost complete decomposition of isoflavone glycosidesoccurred after the reaction at pH 3.5 for 6 hours, and glycitein was100%, genistein 97%, and daidzein 93%. In the case of concentratedsoybean protein, after the reaction at pH 3.7 for 6 hours, glycitein was78%, genistein 69%, and daidzein 75%. In the case of defatted soybean,maximum isolation of aglycons was observed after the reaction at pH 3.4for 6 hours, and glycitein was 68%, genistein 69%, and daidzein 71%.

EXAMPLE 6 Examination of Substrate Concentration in the Conversion intoIsoflavone Aglycons by Diglycosidase Using Soybean Materials

Into 20 mM acetate buffer of pH 4.0 was suspended 0.1 g, 0.25 g, 0.5 g,1.0 g, or 1.5 g of each of various soybean materials (roasted soy flour(manufactured by Fuji Shokuryo K.K.), soymilk (manufactured by GitohShokuhin K.K.), defatted soybean (manufactured by Fuji Seiyu K.K.),concentrated soybean protein (manufactured by Fuji Seiyu K.K.)). The pHof the suspension was measured and the liquid volume was adjusted to 4.5mL while the pH was adjusted to 4.0 with 1N hydrochloric acid. An enzymesolution wherein diglycosidase activity of crude diglycosidase wasadjusted to 1.88 AU/mL was added thereto in an amount of 0.5 mL, wherebythe final liquid volume was 5.0 mL. Namely, the ratio of the soybeanmaterial in the reaction mixture was 2%, 5%, 10%, 20%, or 30% (w/v). Thewhole was reacted at 55° C. under shaking. To 5 mL of the reactionmixture was added 7 mL of ethanol after 0, 1, 3, and 6 hours of thereaction. After ultrasonication, the mixture was thoroughly mixed. Itwas subjected to centrifugal separation at 2,000 rpm and roomtemperature for 5 minutes. Then, 1 μL of the supernatant was placed in a1.5 mL microtube and was subjected to centrifugal separation at 15,000rpm and 4° C. for 10 minutes. The supernatant was filtered through afilter and each sample was suitably diluted by a factor of 1 to 6depending on the substrate concentration. Fifty μL of the dilutedsolution was analyzed by HLPC (Tables 27 to 38).

TABLE 27 Decomposition efficiency of glycitin family in roasted soyflour Substrate Heat Reaction concentration treatment time GlycosideAglycon (%) of enzyme (h) glycitin malonylglycitin acetylglycitinglycitein 2 no 0 50.2 not detected 33.6 16.2 1 not detected not detectednot detected 100.0 3 not detected not detected not detected 100.0 6 notdetected not detected not detected 100.0 yes 6 53.4 not detected 34.412.2 5 no 0 54.8 not detected 33.8 11.4 1 not detected not detected 11.888.2 3 not detected not detected 7.2 92.8 6 not detected not detectednot detected 100.0 yes 6 54.8 not detected 33.8 11.4 10 no 0 70.3 notdetected 24.2 5.5 1 not detected not detected 16.7 83.3 3 not detectednot detected 9.7 90.3 6 not detected not detected not detected 100.0 yes6 55.0 not detected 35.1 9.9 20 no 0 59.6 not detected 31.8 8.6 1 notdetected not detected 22.9 77.1 3 not detected not detected 13.8 86.2 6not detected not detected 10.0 90.0 yes 6 56.0 not detected 35.0 9.1 30no 0 55.9 not detected 35.3 8.8 1 not detected not detected 28.4 71.6 3not detected not detected 24.4 75.6 6 not detected not detected 13.786.3 yes 6 56.9 not detected 34.4 8.7 

TABLE 28 Decomposition efficiency of genistin family in roasted soyflour Substrate Heat Reaction concentration treatment time GlycosideAglycon (%) of enzyme (h) genistin malonylgenistin acetylgenistingenistein 2 no 0 33.2 not detected 44.7 22.1 1 not detected not detected10.6 89.4 3 not detected not detected 5.0 95.0 6 not detected notdetected 1.5 98.5 yes 6 48.4 not detected 45.0 6.7 5 no 0 47.8 notdetected 44.7 7.5 1 not detected not detected 22.1 77.9 3 not detectednot detected 15.2 84.8 6 not detected not detected 9.2 90.8 yes 6 47.8not detected 44.7 7.5 10 no 0 59.5 not detected 38.2 2.3 1 not detectednot detected 33.3 66.7 3 not detected not detected 25.3 74.7 6 notdetected not detected 18.7 81.3 yes 6 49.5 not detected 45.9 4.6 20 no 050.5 not detected 46.1 3.4 1 not detected not detected 44.1 55.9 3 notdetected not detected 35.8 64.2 6 not detected not detected 28.6 71.4yes 6 49.5 not detected 46.2 4.3 30 no 0 49.4 not detected 46.7 3.8 15.7 not detected 46.6 47.7 3 9.6 not detected 41.4 48.9 6 2.0 notdetected 37.8 60.2 yes 6 50.1 not detected 46.1 3.8 

TABLE 29 Decomposition efficiency of daidzin family in roasted soy flourSubstrate Heat Reaction concentration treatment time Glycoside Aglycon(%) of enzyme (h) daidzin malonyldaidzin acetyldaidzin daidzein 2 no 030.2 not detected 47.6 22.3 1 not detected not detected 9.2 90.8 3 notdetected not detected not detected 100.0 6 not detected not detected notdetected 100.0 yes 6 46.6 not detected 47.2 6.2 5 no 0 46.1 not detected46.1 7.7 1 not detected not detected 20.9 79.1 3 not detected notdetected 12.5 87.5 6 not detected not detected 6.8 93.2 yes 6 46.1 notdetected 46.1 7.7 10 no 0 60.1 not detected 38.5 1.4 1 not detected notdetected 30.5 69.5 3 not detected not detected 21.5 78.5 6 not detectednot detected 14.9 85.1 yes 6 47.5 not detected 47.7 4.8 20 no 0 50.3 notdetected 46.1 3.7 1 not detected not detected 40.7 59.3 3 not detectednot detected 32.6 67.4 6 not detected not detected 25.1 74.9 yes 6 48.6not detected 47.2 4.2 30 no 0 47.7 not detected 47.6 4.7 1 not detectednot detected 46.4 53.6 3 not detected not detected 42.6 57.4 6 notdetected not detected 34.3 65.7 yes 6 49.0 not detected 47.4 3.6 

TABLE 30 Decomposition efficiency of glycitin family in soymilk HeatSubstrate treatment Reaction concentration of time Glycoside Aglycon (%)enzyme (h) glycitin malonylglycitin acetyl-glycitin glycitein 2 no 071.8 28.2 not detected not detected 1 not detected not detected notdetected 100.0 3 not detected not detected not detected 100.0 6 notdetected not detected not detected 100.0 yes 6 51.8 48.2 not detectednot detected 5 no 0 49.4 50.6 not detected not detected 1 not detected24.6 not detected 75.4 3 not detected 7.5 not detected 92.5 6 notdetected not detected not detected 100.0 yes 6 56.7 43.3 not detectednot detected 10 no 0 52.5 41.1 not detected 6.4 1 not detected 26.4 notdetected 73.6 3 not detected 10.5 not detected 89.5 6 not detected 4.2not detected 95.8 yes 6 56.8 37.0 not detected 6.2 20 no 0 52.4 41.3 notdetected 6.3 1 not detected 27.8 not detected 72.2 3 not detected 9.4not detected 90.6 6 not detected 5.9 not detected 94.1 yes 6 55.5 38.8not detected 5.7 30 no 0 50.9 43.0 not detected 6.1 1 not detected 33.1not detected 66.9 3 not detected 15.9 not detected 84.1 6 not detected7.7 not detected 92.3 yes 6 55.8 37.5 not detected 6.7 

TABLE 31 Decomposition efficiency of genistin family in soymilkSubstrate Heat Reaction concentration treatment time Glycoside Aglycon(%) of enzyme (h) genistin malonylgenistin acetyl-genistin genistein 2no 0 54.5 35.0 not detected 10.5 1 not detected 15.3 not detected 84.7 3not detected 3.5 not detected 96.5 6 not detected 0.0 not detected 100.0yes 6 38.0 49.5 not detected 12.5 5 no 0 31.5 58.2 not detected 10.3 1not detected 24.9 not detected 75.1 3 not detected 6.9 not detected 93.16 not detected 2.1 not detected 97.9 yes 6 37.9 52.5 not detected 9.7 10no 0 32.9 56.5 0.5 10.1 1 not detected 31.5 not detected 68.5 3 notdetected 11.9 not detected 88.1 6 not detected 4.8 not detected 95.2 yes6 37.6 51.2 0.5 10.6 20 no 0 33.3 56.3 0.6 9.9 1 not detected 37.6 notdetected 62.4 3 not detected 15.7 not detected 84.3 6 not detected 10.7not detected 89.3 yes 6 38.6 51.0 0.6 9.8 30 no 0 33.6 55.9 0.6 9.9 1not detected 42.5 not detected 57.5 3 not detected 25.4 not detected74.6 6 not detected 17.1 not detected 82.9 yes 6 38.6 50.0 0.6 10.9 

TABLE 32 Decomposition efficiency of daidzin family in soymilk SubstrateHeat Reaction concentration treatment time Glycoside Aglycon (%) ofenzyme (h) daidzin malonyldaidzin acetyl-daidzin daidzein 2 no 0 53.532.2 not detected 14.4 1 not detected 27.5 not detected 72.5 3 notdetected 9.9 not detected 90.1 6 not detected 2.9 not detected 97.1 yes6 39.8 47.0 not detected 13.1 5 no 0 34.2 53.6 not detected 12.2 1 notdetected 34.2 not detected 65.8 3 not detected 15.9 not detected 84.1 6not detected 6.1 not detected 93.9 yes 6 40.5 47.6 not detected 12.0 10no 0 34.6 53.4 not detected 12.0 1 not detected 37.4 not detected 62.6 3not detected 20.5 not detected 79.5 6 not detected 10.7 not detected89.3 yes 6 39.4 47.8 not detected 12.8 20 no 0 34.1 53.6 not detected12.3 1 not detected 41.2 not detected 58.8 3 not detected 21.5 notdetected 78.5 6 not detected 16.4 not detected 83.6 yes 6 39.8 48.1 notdetected 12.1 30 no 0 34.9 53.5 not detected 11.6 1 not detected 43.8not detected 56.2 3 not detected 29.7 not detected 70.3 6 not detected21.9 not detected 78.1 yes 6 40.0 46.9 not detected 13.1 

TABLE 33 Decomposition efficiency of glycitin family in concentratedsoybean protein Substrate Heat Reaction concentration treatment timeGlycoside Aglycon (%) of enzyme (h) glycitin malonylglycitinacetylglycitin glycitein 2 no 0 52.2 0.4 35.8 11.6 1 not detected 0.414.3 85.3 3 not detected 0.2 5.3 94.4 6 not detected not detected 0.0100.0 yes 6 53.4 0.4 35.0 11.2 5 no 0 52.9 0.4 35.6 11.2 1 not detected0.5 27.1 72.4 3 not detected 0.4 17.8 81.9 6 not detected 0.2 12.3 87.4yes 6 54.0 0.4 34.4 11.3 10 no 0 52.9 0.3 35.7 11.0 1  1.6 0.4 31.2 66.83 not detected 0.3 26.2 73.5 6 not detected 0.3 23.9 75.8 yes 6 53.4 0.335.2 11.1 20 no 0 53.1 0.2 35.9 10.8 1 13.2 0.3 35.4 51.2 3 not detected0.2 33.6 66.2 6 not detected 0.3 30.5 69.2 yes 6 52.8 0.4 35.8 11.1 30no 0 57.5 0.4 33.9 8.2 1  2.0 0.6 35.9 61.5 3 not detected 0.5 30.7 68.96 not detected 0.3 28.3 71.4 yes 6 58.0 0.4 33.4 8.2 

TABLE 34 Decomposition efficiency of genistin family in concentratedsoybean protein Substrate Heat Reaction concentration treatment timeGlycoside Aglycon (%) of enzyme (h) genistin malonylgenistinacetylgenistin genistein 2 no 0 50.5 not detected 42.9 6.6 1  1.2 notdetected 20.0 78.7 3 not detected not detected 8.3 91.7 6 not detectednot detected 3.3 96.7 yes 6 51.6 not detected 41.3 7.0 5 no 0 51.2 notdetected 43.1 5.8 1 not detected not detected 36.4 63.6 3 not detectednot detected 26.3 73.7 6 not detected not detected 17.8 82.2 yes 6 52.2not detected 41.7 6.2 10 no 0 50.6 not detected 43.4 5.9 1  1.3 notdetected 42.8 55.8 3 not detected not detected 37.3 62.7 6 not detectednot detected 33.2 66.8 yes 6 50.9 not detected 42.8 6.3 20 no 0 50.4 notdetected 43.7 5.9 1  6.0 not detected 47.0 46.9 3 not detected notdetected 44.6 55.4 6 not detected not detected 42.3 57.7 yes 6 50.8 notdetected 43.2 6.1 30 no 0 54.6 not detected 43.5 1.9 1  2.8 not detected65.3 31.9 3 not detected not detected 61.0 39.0 6 not detected notdetected 55.9 44.1 yes 6 55.6 not detected 42.4 2.0 

TABLE 35 Decomposition efficiency of daidzin family in concentratedsoybean protein Substrate Heat Reaction concentration treatment timeGlycoside Aglycon (%) of enzyme (h) diadzin malonydaidzin acetly-daidzindaidzein 2 no 0 52.8 not detected 44.0 3.2 1 not detected not detected17.8 82.2 3 not detected not detected  5.3 94.7 6 not detected notdetected not detected 100.0 yes 6 52.6 not detected 43.2 4.2 5 no 0 53.0not detected 44.0 3.1 1 not detected not detected 32.6 67.4 3 notdetected not detected 20.5 79.5 6 not detected not detected 12.0 88.0yes 6 53.5 not detected 43.0 3.5 10 no 0 52.8 not detected 44.2 3.0 1 2.2 not detected 39.7 58.1 3 not detected not detected 32.9 67.1 6 notdetected not detected 26.9 73.1 yes 6 53.3 not detected 43.4 3.3 20 no 052.9 not detected 44.1 3.0 1  0.7 not detected 46.4 52.9 3 not detectednot detected 41.5 58.5 6 not detected not detected 37.9 62.1 yes 6 52.8not detected 44.0 3.2 30 no 0 49.2 not detected 49.2 1.7 1  2.7 notdetected 56.6 40.7 3 not detected not detected 49.9 50.1 6 not detectednot detected 42.8 57.2 yes 6 49.8 not detected 48.4 1.8 

TABLE 36 Decomposition efficiency of glycitin family in defatted soybeanSubstrate Heat Reaction concentration treatment time Glycoside Aglycon(%) of enzyme (h) glycitin malonylglycitin acetylglycitin glycitein 2 no0 not detected not detected not detected not detected 1 not detected notdetected not detected not detected 3 not detected not detected notdetected not detected 6 not detected not detected not detected notdetected yes 6 not detected not detected not detected not detected 5 no0 not detected 58.4 not detected 41.6 1 not detected 41.6 not detected58.4 3 not detected 31.8 not detected 68.2 6 not detected 19.5 notdetected 80.5 yes 6 not detected 58.5 not detected 41.5 10 no 0 notdetected 53.0 not detected 47.0 1 not detected 54.7 not detected 45.3 3not detected 36.6 not detected 63.4 6 not detected 27.9 not detected72.1 yes 6 not detected 57.6 not detected 42.4 

TABLE 37 Decomposition efficiency of genistin family in defatted soybeanSubstrate Heat Reaction concentration treatment time Glycoside Aglycon(%) of enzyme (h) genisitin malonylgenistin acetylgenistin genistein 2no 0 37.7 45.4 1.8 15.1 1 not detected 31.4 not detected 68.6 3 notdetected 17.0 not detected 83.0 6 not detected 11.4 not detected 88.6yes 6 38.7 41.8 1.5 18.0 5 no 0 40.0 43.9 1.9 14.2 1 not detected 38.00.8 61.2 3 not detected 29.6 0.5 69.9 6 not detected 19.0 0.3 80.7 yes 641.8 41.1 1.8 15.4 10 no 0 42.4 41.6 1.9 14.1 1 not detected 41.5 1.357.2 3 not detected 35.1 0.9 64.0 6 not detected 29.1 0.6 70.3 yes 644.3 39.4 1.7 14.5 

TABLE 38 Decomposition efficiency of daidzin family in defatted soybeanSubstrate Heat Reaction concentration treatment time Glycoside Aglycon(%) of enzyme (h) daidzin malonyldaidzin acetyldaidzin daidzein 2 no 042.3 42.2 not detected 15.5 1 not detected 35.2 not detected 64.8 3 notdetected 21.2 not detected 78.8 6 not detected 16.5 not detected 83.5yes 6 43.7 38.1 not detected 18.2 5 no 0 44.0 41.1 not detected 14.9 1not detected 37.7 not detected 62.3 3 not detected 30.6 not detected69.4 6 not detected 21.0 not detected 79.0 yes 6 45.8 38.1 not detected16.1 10 no 0 45.6 39.7 not detected 14.7 1 not detected 39.5 notdetected 60.5 3 not detected 34.2 not detected 65.8 6 not detected 29.0not detected 71.0 yes 6 47.5 37.0 not detected 15.5

By combining maximum reaction temperature and pH, in all the materialsexamined, isolation of each isoflavone glucoside was found to be 70% ormore when the material concentration is 10% or less. In particular, whenthe material concentration ranges from 2% to 5%, it was revealed thatalmost 100% conversion of isoflavone glycosides into aglycons occurred.

EXAMPLE 7 Influence of Commercially Available Enzyme PreparationsAccelerating the Conversion into Aglycon Isoflavones by Diglycosidase

Soyaflavone (manufactured by Fuji Seiyu K.K.) was suspended into 1.0 Msodium acetate of pH 3.0 and the substrate concentration was adjusted to30% (w/v) and pH of the solution to 5.0. The suspension waspre-incubated at 50° C. for 1 hour, whereby the temperature of thesuspension was elevated to 50° C. To the suspension was added eachcommercially available enzyme preparation (Amylase AD “Amano” 1, YL-15,Gluczyme NL4.2, Transglucosidase L “Amano”, all manufactured by AmanoEnzyme Inc.) solely or in combination with diglycosidase (0.3 AU) so asto be 0.1% (w/v). The whole was reacted at 50° C. for 6 hours and thechange of the composition of isoflavone glycosides and aglyconisoflavones was analyzed by HPLC. Isoflavone glycosides and aglyconisoflavones were quantitatively determined. Among them, relative valuesof the aglycon isoflavones were shown in FIG. 1, ideal values of theaglycon isoflavones being 100%.

The ideal value of each aglycon isoflavone was calculated as followsbased on the content of each isoflavone glycoside and aglycon isoflavonein the case that no enzyme was added.Ideal value of aglycon isoflavone=AG+G1×M _(AG) /M _(G1) +G2×M _(AG) /M_(G2)+ . . .AG; amount of aglycon isoflavone, G1; amount of isoflavone glycoside,M_(AG); molecular weight of aglycon isoflavone, M_(G); molecular weightof isoflavone glycoside

Each commercially available enzyme preparation it self could hardlyhydrolyze glycosides but the combination with diglycosidase obviouslyincreased the amount of isolated aglycon isoflavones. For example, thecombination of diglycosidase with Amylase AD “Amano” 1 resulted in2.1-fold increase of glycitein and 1.3-fold increase of genistein ascompared with the action of diglycosidase alone. However, about 1.1-foldincrease was observed for daidzein. It was revealed that the combinationof diglycosidase with YL-15 resulted in 1.8-fold increase of glycitein,1.2-fold increase of genistein, and 1.1-fold increase of daidzein, thecombination of diglycosidase with Gluczyme NL-4.2 resulted in 0.8-foldincrease of glycitein, 1.1-fold increase of genistein, and 1.0-foldincrease of daidzein, and the combination of diglycosidase withTransglucosidase L “Amano” resulted in 1.6-fold increase of glycitein,1.0-fold increase of genistein, and 1.0-fold increase of daidzein.

EXAMPLE 8 Examination of Effective Amount of Amylase AD “Amano” 1Accelerating the Conversion into Aglycon Isoflavones by Diglycosidase

Roasted soy flour (manufactured by Fuji Shokuhin K.K.) was suspendedinto 0.1 M sodium acetate adjusted to pH 3.15, the substrateconcentration was adjusted to 30% (w/v), and the pH of the solution to5.0. The suspension was pre-incubated at 50° C. for 1 hour, whereby thetemperature of the suspension was elevated to 50° C. To the suspensionwere added diglycosidase so as to be a final concentration of 0.1% (w/v)(0.3 AU) and Amylase AD “Amano” 1 (manufactured by Amano Enzyme Innc.)so as to be 0.1, 0.05, 0.01, 0.005, and 0.0001% (w/v). The whole wasreacted at 50° C. for 3 hours and the change of isoflavone compositionwas analyzed by HPLC.

As shown in FIG. 2, it is found that the isolated amount of aglyconrather increased at a concentration lower than 0.1% (w/v). An effect wasobserved even at a mall amount of 0.0001% (w/v). At 0.005% (w/v),glycitein and genistein increased by a factor of 1.23 and 1.20,respectively. In the case of daidzein, 1.2-fold increase was observed.By the way, 0% means the results in the case of diglycosidase alone.

EXAMPLE 9 Flavor Improvement of Soybean Protein by Diglycosidase or EachCommercially Available Enzyme Preparation

To 10 g of Fujipro (separated soybean protein, manufactured by FujiSeiyu K.K.) was added 90 mL of water, and the whole was thoroughly mixedto obtain a soybean protein solution. Thereto was added diglycosidase oreach of commercially available enzyme preparation (Amylase AD “Amano” 1,ADG-S-DS, Lipase A “Amano” 6, Lactase F-DS, Lactase F, Cellulase A“Amano” 3, Hemicellulase “Amano” 90G, Protease B, YL-15, Pectinase PL“Amano”, Transglucosidase L “Amano”, Gluczyme NL4.2, all manufactured byAmano Enzyme Inc.) so that diglycosidase (0.5 AU) or each enzymepreparation was contained in the soybean protein solution in aconcentration of 0.25% (w/v). The treatment was carried out at 50° C.for 5 hours. By the way, the reaction pH was 7.1. For a sensory test, pHwas not adjusted for avoiding the influence of a buffer (the same shallapply to Examples 10 to 12).

Sensory Test (Flavor Improvement of Separated Soybean Protein Treatedwith Diglycosidase or Each Commercially Available Enzyme Preparation)

For carrying out a sensory test, each soybean protein treated withdiglycosidase or each commercially available enzyme preparation wassubjected to centrifugal separation at 1500×g and 4° C. for 20 minutes.The precipitate was removed and pH of the supernatant was adjusted to6.0 with hydrochloric acid. Using a solution obtained by two-folddilution of the solution, the test was carried out. The evaluation wasconducted by five expert panelists, and the deliciousness,bitterness·astringency, and aftertaste were compared with those ofControl untreated with the enzyme (Table 39).

TABLE 39 Flavor-improving effect of enzyme preparation on separatedsoybean protein Panelist A Panelist B Panelist C bitterness · bitterness· bitterness · Enzyme name deliciousness astringency aftertastedeliciousness astringency aftertaste deliciousness astringencyaftertaste Untreated (Control) ± ± ± ± ± ± ± ± ± Diglycosidase ± −− + ±− + ± −− ± Amylase AD ± −− + ± −− ± ± − ± “Amano” 1 ADG-D-DS ± −− + ± −± ± − ++ Lipase A “Amano” 6 ± ± + ± − + ± ± + Lactase F-DS ± ± ± ± ± − ±± ± Lactase F “Amano” ± ± ± ± ± ± ± ± ± Cellulase A ± −− + ± −− ± ± − ±“Amano” 3 Hemicellulase ± −− + ± −− ± ± − ± “Amano” 90G Protease B ± + ±± ± ± ± ± ± YL-15 ± +++ + ± +++ ± ± +++ ± Pectinase PL ± ± + ± ± ± ± ± ±Trans- glucosidase L ± ± ± ± ± ± ± ± ± Gluczyme ± ± ± ± ± ± ± ± ±Panelist D Panelist E bitterness · bitterness · Enzyme namedeliciousness astringency aftertaste deliciousness astringencyaftertaste Untreated (Control) ± ± ± ± ± ± Diglycosidase + ± + + − ±Amylase AD “Amano” 1 ± ± ± ± − ± ADG-D-DS + ± + ± − ± Lipase A “Amano” 6± ± + ± ± ± Lactase F-DS ± ± − ± ± ± Lactase F “Amano” ± ± ± ± ± ±Cellulase A “Amano” 3 + − ± + −− ± Hemicellulase “Amano” 90G ± − ± ± ± +Protease B ± ± ± ± ± ± YL-15 ± +++ ± ± + ± Pectinase PL ± ± ± ± ± ±Transglucosidase L ± ± ± ± ± ± Gluczyme ± ± ± ± ± ± ±; No change isobserved as compared with Control. +; This means that “deliciousness”and “aftertaste” are improved and “bitterness·astringency” isstrengthened. −; This means that “deliciousness” and “aftertaste” becomeworse and “bitterness·astringency” is reduced. The increase in numberof + and − means that the tendency becomes strong.

As a result, the bitterness-astringency was reduced or disappeared bydiglycosidase, Amylase AD “Amano” 1, ADG-S-DS, Lipase A “Amano” 6,Cellulase A “Amano” 3, or Hemicellulase “Amano” 90G.

Also, it was revealed that the treatment with diglycosidase, Amylase AD“Amano” 1, ADG-S-DS, Lipase A “Amano” 6, Cellulase A “Amano” 3,Hemicellulase “Amano” 90G, YL-15, or Pectinase PL “Amano” was effectivefor improving aftertaste. It was revealed that these enzyme preparationsimprove overall flavor, for example, appearance of sweetness andreduction of smelling of grass, other than the examined articles.

EXAMPLE 10 Flavor Improvement of Soybean Protein by Diglycosidase andEach Commercially Available Enzyme Preparation

To 10 g of Fujipro (separated soybean protein, manufactured by FujiSeiyu K.K.) was added 90 mL of water, and the whole was thoroughly mixedto obtain a soybean protein solution. Thereto was added diglycosidaseand each of commercially available enzyme preparation (Amylase AD“Amano” 1, ADG-S-DS, Lipase A “Amano” 6, Lactase F-DS, Lactase F,Cellulase A “Amano” 3, Hemicellulase “Amano” 90G, Protease B, YL-15,Pectinase PL “Amano”, Transglucosidase L “Amano”, Gluczyme NL4.2, allmanufactured by Amano Enzyme Inc.) so that diglycosidase was contained6.5 AU in the soybean protein solution and each enzyme preparation in aconcentration of 0.25% (w/v). The treatment was carried out at 50° C.for 5 hours to obtain an enzyme-treated soybean protein. By the way, thereaction pH was 7.1.

Sensory Test (Flavor Improvement of Separated Soybean Protein Treatedwith Diglycosidase and Each Commercially Available Enzyme Preparation)

For carrying out a sensory test, each soybean protein treated withenzymes was subjected to centrifugal separation at 1500×g and 4° C. for20 minutes. The precipitate was removed and pH of the supernatant wasadjusted to 6.0 with hydrochloric acid. Using a solution obtained bytwo-fold dilution of the solution, the test was carried out. Theevaluation was conducted by five expert panelists, and thedeliciousness, bitterness·astringency, and aftertaste were compared withthose of Control untreated with the enzyme (Table 40).

TABLE 40 Flavor improvement of separated soybean protein by combinationof enzymes Panelist A Panelist B Panelist C bitterness · bitterness ·bitterness · Enzyme name deliciousness astringency aftertastedeliciousness astringency aftertaste deliciousness astringencyaftertaste Untreated (Control) ± ± ± ± ± ± ± ± ± Diglycosidase + Amylase± −−− + + −−− + ± −− ± AD “Amano” 1 Diglycosidase + ADG-S- + −−− ± + − ±++ −− ++ DS Diglycosidase + Lipase + −−− ± ± − ± + ± + A “Amano”6Diglycosidase + Lactase ± − ± ± − ± + ± ± F-DS Diglycosidase + Lactase ±± + + − ± + ± + F “Amano” Diglycosidase + ± −−− ++ + −−− + + − ±Cellulase A “Amano”3 Diglycosidase + ± −−− ++ + −− ± + − ± Hemicellulase“Amano”90G Diglycosidase + ± −−− ± + − ± ± −− ± Protease BDiglycosidase + YL-15 ± + + + + + + ± + Diglycosidase + ± − ++ + − ± +−− ± Pectinase PL “Amano” Diglycosidase + ± − ± ± − ± ± − ±Transglucosidase L “Amano” Diglycosidase + ± ± ± + − ± ± ± ± GluczymeNL4.2 Panelist D Panelist E bitterness · bitterness · Enzyme namedeliciousness astringency aftertaste deliciousness astringencyaftertaste Untreated (Control) ± ± ± ± ± ± Diglycosidase + Amylase AD“Amano”1 ++ ± ± + −− ++ Diglycosidase + ADG-S-DS ++ − ++ ± −− +Diglycosidase + Lipase A “Amano”6 ++ ± + ± − + Diglycosidase + LactaseF-DS ± ± ± + − ± Diglycosidase + Lactase F “Amano” + ± + ± −− ++Diglycosidase + Cellulase A “Amano”3 ++ − ± + −−− + Diglycosidase +Hemicellulase “Amano” 90G ++ − ++ + − ± Diglycosidase + Protease B ++ ±± ± − + Diglycosidase + YL-15 + ++ + ± ± + Diglycosidase + Pectinase PL“Amano” ++ ± + ± − + Diglycosidase + Transglucosidase L “Amano” + ± ± +− ± Diglycosidase + Gluczyme NL4.2 + ± ± ± − ±

As a result, it was revealed that effects, for example, appearance ofsweetness, reduction of bitterness·astringency, or improvement ofaftertaste were observed in all the combinations of diglycosidase andeach commercially available enzyme preparation (diglycosidase andAmylase AD “Amano” 1, diglycosidase and ADG-S-DS, diglycosidase andLipase A “Amano” 6, diglycosidase and Lactase F-DS, diglycosidase andLactase F “Amano”, diglycosidase and Cellulase A “Amano” 3,diglycosidase and Hemicellulase “Amano” 90G, diglycosidase and ProteaseB, diglycosidase and YL-15, diglycosidase and Pectinase PL “Amano”,diglycosidase and Transglucosidase L “Amano”, diglycosidase and GluczymeNL4.2) shown in the table. From these facts, it was evident thatflavor-improving effect was stronger in the combination of diglycosidaseand each commercially available enzyme preparation than in the case ofdiglycosidase alone.

EXAMPLE 11 Flavor Improvement of Soymilk by Diglycosidase or EachCommercially Available Enzyme Preparation

To 20 mL of an ingredient-unadjusted soymilk (Gitoh Shokuhin K.K.) wasadded diglycosidase or each of commercially available enzyme preparation(ADG-S-DS, Amylase AD “Amano” 1, Cellulase A “Amano” 3, Hemicellulase“Amano” 90G, all manufactured by Amano Enzyme Inc.) so thatdiglycosidase was contained 6.5 AU in the soymilk or each enzymepreparation in a concentration of 0.25% (w/v). The treatment was carriedout at 55° C. for 1.5 hours. Thereafter, the enzymes were inactivated byheat treatment at 70° C. for 1 hour. By the way, the reaction pH was6.6.

Sensory Test (Flavor Improvement of Soymilk Treated with Diglycosidaseor Each Commercially Available Enzyme Preparation)

The enzyme-treated liquid thus obtained was diluted with water by afactor of 3, and then subjected to a sensory test. The evaluation wasconducted by five expert panelists, and the deliciousness, bitternessastringency, and aftertaste were compared with those of Controluntreated with the enzyme (Table 41).

TABLE 41 Flavor-improving effect of enzyme preparation on soymilkPanelist A Panelist B Panelist C bitter- bitter- bitter- ness* ness*ness* Enzyme sweet- astrin- after- sweet- astrin- after- sweet- astrin-after- name ness gency taste ness gency taste ness gency taste Untreated± ± ± ± ± ± ± ± ± (Control) Diglycosidase ± − ± ± ± ± + + − + ADG-D-DS +− ± + − + ± − ± Amylase AD + − − + + − + + − ± “Amano” 1 Cellulase A ± ±± + − + ± − ± “Amano” 3 Hemicellulase ± ± ± + − + ± ± ± “Amano” 90GPanelist D Panelist E bitter- bitter- ness* ness* Enzyme sweet- astrin-after- sweet- astrin- after- name ness gency taste ness gency tasteUntreated ± ± ± ± ± ± (Control) Diglycosidase + ± ± + ± ± ADG-D-DS + ±± + ± ± Amylase AD ± ± ± ± ± ± “Amano” 1 Cellulase A ± ± ± + − + “Amano”3 Hemicellulase ± ± ± + − + “Amano” 90G

It was revealed that appearance of sweetness, reduction ofbitterness·astringency, and improvement of aftertaste were effected bydiglycosidase or a commercially available enzyme preparation.

EXAMPLE 12 Flavor Improvement of Soymilk by Diglycosidase and EachCommercially Available Enzyme Preparation

To 20 mL of an ingredient-unadjusted soymilk (Gitoh Shokuhin K.K.) wasadded diglycosidase and each of commercially available enzymepreparation (ADG-S-DS, Amylase AD “Amano” 1, Cellulase A “Amano” 3, orHemicellulase “Amano” 90G, all manufactured by Amano Enzyme Inc.) sothat diglycosidase was contained 6.5 AU in the soymilk and each enzymepreparation in a concentration of 0.25% (w/v). The treatment was carriedout at 55° C. for 1.5 hours. Thereafter, the enzymes were inactivated byheat treatment at 70° C. for 1 hour. By the way, the reaction pH was6.6.

Sensory Test (Flavor Improvement of Soymilk Treated with Diglycosidaseand Each Commercially Available Enzyme Preparation)

The enzyme-treated liquid thus obtained was -diluted with water by afactor of 3, and then subjected to a sensory test. The evaluation wasconducted by five expert panelists, and the deliciousness,bitterness·astringency, and aftertaste were compared with those ofControl untreated with the enzyme (Table 42).

TABLE 42 Flavor improvement of soymilk by combination of enzymesPanelist A Panelist B Panelist C bitter- bitter- bitter- ness* ness*ness* Enzyme sweet- astrin- after- sweet- astrin- after- sweet- astrin-after- name ness gency taste ness gency taste ness gency taste Untreated± ± ± ± ± ± ± ± ± (Control) Diglycosidase + + − − ± + − + + + − − +ADG-S-DS Diglycosidase + + − − − + + − − ± + − − ± Amylase AD “Amano” 1Diglycosidase + ± − ± + − + + + + − − + Cellulase A “Amano” 3Diglycosidase + + − ± + − + + − ± Hemicellulase “Amano” 90G Panelist DPanelist E bitter bitter- ness* ness* Enzyme sweet- astrin- after-sweet- astrin- after- name ness gency taste ness gency taste Untreated ±± ± ± ± ± (Control) Diglycosidase + + − ± + + ± ± ADG-S-DSDiglycosidase + + − + + ± ± Amylase AD “Amano” 1 Diglycosidase + +− + + + − + Cellulase A “Amano” 3 Diglycosidase + + ± ± + + − +Hemicellulase “Amano” 90G

As a result of the sensory test, it was revealed that effects, forexample, appearance of sweetness, reduction of bitterness·astringency,or improvement of aftertaste were observed in all the combinations ofdiglycosidase and each commercially available enzyme preparation(diglycosidase and ADG-S-DS, diglycosidase and Amylase AD “Amano” 1,diglycosidase and Cellulase A “Amano” 3, diglycosidase and Hemicellulase“Amano” 90G) shown in the table. Furthermore, it was revealed that sucha treatment with the enzymes improves overall flavor, for example,reduction of smelling of grass. From these facts, it was evident thatflavor-improving effect was stronger in the combination of diglycosidaseand each commercially available enzyme preparation than in the case ofdiglycosidase alone or a commercially available enzyme preparationalone.

EXAMPLE 13 Formation of Aglycon Isoflavones from Defatted SoybeanProtein by Diglycosidase at the pH in a Stomach

Into 20 mM acetate buffer of pH 4.0 was suspended 0.05 g of defattedsoybean protein (Fuji Seiyu K.K.). The pH of the suspension was measuredand the liquid volume was adjusted to 4.5 mL while the pH was adjustedto 4.0 with 1N hydrochloric acid. An enzyme solution wherein thediglycosidase activity of crude diglycosidase was adjusted to 1.88 AU/mLwas added thereto in an amount of 0.5 mL, whereby the final liquidvolume was 5.0 mL (concentration of defatted soybean protein: 1% (w/v)),followed by treatment at 37° C. for 3 hours. After the treatment, 75 μLof methanol and 500 μL of water were added to 25 μL of the reactionmixture. The whole was filtered through a 0.2 μm filter and then thefiltrate was further diluted with water by a factor of 2.5, followed byHPLC analysis.

As a result of treating defatted soybean protein at pH 4 which was thepH range in a stomach during a meal as described above, no isolation ofaglycon isoflavones was observed in the treatment at pH 4 without addingthe enzyme, but isolation of aglycon isoflavones was observed in theproduct treated with diglycosidase. Therefore, it was proved thatdiglycosidase could convert isoflavone glycosides into aglyconisoflavones under the pH condition in a stomach.

EXAMPLE 14 Formation of Aglycon Isoflavones from Roasterd Soy Flour byDiglycosidase at the pH in a Stomach

Into 100 mL of 50 mM acetate buffer (pH 5) was suspended 2.5 g ofroasted soy flour (manufactured by Kakudai Sangyo).

To 3 mL of the suspension was added 0.001, 0.002, 0.005, 0.01, 0.025,0.05, 0.075, 0.15, 0.374, 0.75, or 1.5 mg of diglycosidase (290 AU/g),followed by incubation under shaking at 37° C. for 30 minutes. After thereaction, 10 mL of methanol was added thereto, and aglycon isoflavoneswere extracted and analyzed by HPLC.

As described above, the formation of aglycon isoflavones from isoflavoneglycosides contained in soy flour was investigated after the reaction atpH 5 and 37° C. for 30 minutes on the assumption of the environment in astomach. As a result, as shown in Table 43 and FIG. 3, 80% or more ofthe isoflavone glycosides could be converted into aglycon isoflavones.This result suggests that aglycon isoflavones may be formed fromisoflavone glycosides in a stomach when diglycosidase is orallyadministered.

TABLE 43 Relationship between added amount of diglycosidase andformation (%) of aglycon isoflavones aglycon isoflavones diglycosidase(%) 1.500 86.1% 0.750 81.3% 0.374 74.7% 0.150 65.4% 0.075 56.3% 0.05051.1% 0.025 44.3% 0.010 31.0% 0.005 20.3% 0.002 12.8% 0.001  9.2% 0 5.0%

EXAMPLE 15 Formation of Aglycon Isoflavones from an IsoflavonePreparation by Diglycosidase at the pH in a Stomach

Into 100 mL of 50 mM acetate buffer (pH 5) was suspended 150 mg of anisoflavone preparation (manufactured by Nature's Bountry, USA). To 3 mLof the suspension was added 0, 0.00045, 0.00113, 0.00225, 0.0045, 0.009,0.0225, 0.045, 0.09, 0.18, 0.45, or 0.9 mg of diglycosidase (290 AU/g),followed by incubation under shaking at 37° C. for 30 minutes. After thereaction, 10 mL of methanol was added thereto, and aglycon isoflavoneswere extracted and analyzed by HPLC.

As described above, the formation of aglycon isoflavones from isoflavoneglycosides contained in the isoflavone preparation was investigatedafter the reaction at pH 5 and 37° C. for 30 minutes on the assumptionof the environment in a stomach. As a result, as shown in Table 44 andFIG. 4, 80% or more of the isoflavone glycosides could be converted intoaglycon isoflavones. This result suggests that aglycon isoflavones maybe formed from isoflavone glycosides in a stomach when diglycosidase isorally administered.

TABLE 44 Relationship between added amount of diglycosidase andformation (%) of aglycon isoflavones aglycon diglycosidase isoflavones(mg) (%) 0.90000 79.4% 0.45000 78.7% 0.18000 75.3% 0.09000 70.7% 0.0450064.6% 0.02250 59.2% 0.00900 47.5% 0.00450 38.9% 0.00225 30.3% 0.0011325.6% 0.00045 21.5% 0.00000 20.0%

INDUSTRIAL APPLICABILITY

A physiologically active substance of aglycon type can be efficientlyproduced, without resort to any acid/alkali treatment or fermentationand substantially without changing the physical properties of amaterial.

Since diglycosidase has a nature of well acting on 6″-O-acetyl and6″-O-malonylglucosides which are resistant to decomposition byconventional glucosidase, the process can be conducted at one-stepwithout requiring the process of converting decomposition-resistantisoflavone glycosides into isoflavone glycosides decomposable byglucosidase, the process being described in JP-A-10-117792. Moreover,the present process hardly causes change of physical properties of astarting material derived from decomposition of proteins orphospholipids, the decomposition being caused during the process ofhydrolysis with a strong acid. Furthermore, by using diglycosidaseand/or a specific enzyme preparation, the aglycon content in a proteinor protein-containing food can be increased and also the flavor thereofcan be improved.

1. A process for producing an aglycon which comprises forming an aglyconby treating, with diglycosidase, a glycoside containing an isoflavone asthe aglycon, wherein said diglycosidase has activity to act upon adisaccharide glycoside to release saccharides in a disaccharide unit,wherein said diglycosidase is isolated from a micoroorganism, whereinsaid diglycosidase is not inhibited by free glucose, wherein saiddiglycosidase has an optimum temperature of about 55° C., wherein saiddiglycosidase has an optimum pH of about 3.5–5, and wherein thediglycosidase is diglycosidase produced by Penicillium multicolor IAM7153 or a mutant strain thereof.
 2. The process for producing an aglyconaccording to claim 1, wherein the glycoside containing an isoflavone asthe aglycon is one or more selected from the group consisting ofdaidzin, genistin, or glycitin and acetyl derivatives, succinylderivatives, or malonyl derivatives thereof.
 3. The process forproducing an aglycon according to claim 1, wherein the diglycosidase isdiglycosidase produced by Penicillium multicolor IAM
 7153. 4. A methodof converting a physiologically active substance of glycoside type intoa physiologically active substance of aglycon type, which comprisestreating the physiologically active substance of glycoside type withdiglycosidase, wherein said physiologically active substance ofglycoside type comprises an isoflavone as the aglycon, wherein saiddiglycosidase has activity to act upon a disaccharide glycoside torelease saccharides in a disaccharide unit, wherein said diglycosidaseis isolated from Penicillium multicolor IAM 7153 or a mutant strainthereof, wherein said diglycosidase is not inhibited by free glucose,wherein said diglycosidase has an optimum temperature of about 55° C.,and wherein said diglycosidase has an optimum pH of about 3.5–5.
 5. Aprocess for producing a composition rich in a phytogenic physiologicallyactive substance of aglycon type, which comprises treating a phytogenicmaterial containing a phytogenic physiologically active substance ofglycoside type with diglycosidase, wherein the phytogenicphysiologically active substance of glycoside type comprises anisoflavone as the aglycon, wherein said diglycosidase has activity toact upon a disaccharide glycoside to release saccharides in adisaccharide unit, wherein said diglycosidase is isolated fromPenicillium multicolor IAM 7153 or a mutant strain thereof, wherein saiddiglycosidase is not inhibited by free glucose, wherein saiddiglycosidase has an optimum temperature of about 55° C., and whereinsaid diglycosidase has an optimum pH of about 3.5–5.
 6. A process forproducing an aglycon which comprises forming an aglycon by treating,with diglycosidase, a glycoside containing an isoflavone as the aglycon,wherein said diglycosidase has activity to act upon a disaccharideglycoside to release saccharides in a disaccharide unit, and whereinsaid diglycosidase is prepared by a process comprising: a) culturingPenicillium multicolor IAM 7152 or a mutant strain thereof in a nutrientmedium under aerobic conditions with a pH from 3–8 to effect productionof said diglycosidase in a culture mixture; b) isolating saiddiglycosidase by a combination of centrifugation, ultrafiltrationconcentration, and salting out of said diglycosidase by 50 to 80%ammonium sulfate from said culture mixture to produce an isolatediglycosidase fraction; and c) purifying said diglycosidase from saidisolated diglycosidase fraction by hydrophobic chromatography.